Quality control for reporter screening assays

Abstract

Described herein is a method of quality assurance for high throughput screening systems that uses control nucleic acids to detect and diagnose sample loss, contamination, and suboptimal process steps.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 292,841, filed December 22, 2021, the entirety of which is incorporated herein by reference. [Background technology]

[0002] High throughput screening (HTS) may involve multiple steps that may not function optimally under certain conditions. Inefficiencies may result from imperfections in liquid handling, suboptimal lysis steps, suboptimal enzymatic reactions (including DNase digestion, reverse transcription, amplification, and sequencing), and contamination. Described herein are methods and process steps to manage inefficiencies throughout the high throughput screening process. Summary of the Invention

[0003] High-throughput screening (HTS) has the potential to reveal much about the biology of cells and accelerate the development of new therapeutic molecules. However, HTS as an end-to-end process is fraught with uncertainty because the most useful assays (e.g., those that deploy multiple orthogonal readouts) are subject to variation and data loss during sample processing and data acquisition. The methods described herein enable researchers to generate more reliable data and filter out data of questionable reliability.

[0004] Described herein is a method for improving the data output of high-throughput screening assays.This method is most useful for the assays that use next-generation sequencing as readout, because such assays have multiple steps that can lead to multiple sources of variation, contamination, and / or data loss.The method described herein includes using control nucleic acid that is added to the end-to-end process at various steps, allowing researchers to identify and exclude unreliable data.

[0005] Described herein is a method of quality assurance for a high throughput screening system, the method comprising: (a) providing a first composition comprising a plurality of cells; (b) lysing the plurality of cells to obtain a lysed cell composition, the lysed cell composition comprising a first plurality of control nucleic acids and a second plurality of control nucleic acids, or the first plurality of control nucleic acids and the second plurality of control nucleic acids; (c) preparing a reverse transcription reaction comprising at least a portion of the lysed cell composition and a third plurality of control nucleic acids, a fourth plurality of control nucleic acids, or the third plurality of control nucleic acids and the fourth plurality of control nucleic acids; (d) reverse transcribing the reverse transcription reaction to obtain reverse transcribed nucleic acids; (e) preparing a sequencing library comprising the reverse transcribed nucleic acids; and (f) performing a sequencing reaction on the sequencing library to obtain sequence information for one or more of (i) a plurality of reporters, and (ii) the first plurality of control nucleic acids, the second plurality of control nucleic acids, the third plurality of control nucleic acids, or the fourth plurality of control nucleic acids. In certain embodiments, the method further comprises contacting the lysed cell composition with a DNA degrading agent. In certain embodiments, the method further comprises contacting the lysed cell composition with a fifth plurality of control nucleic acids before contacting the lysed cell composition with the DNA degrading agent. In certain embodiments, the method further comprises contacting the plurality of cells with a test agent. In certain embodiments, the step of contacting the plurality of cells with the test agent is prior to step (b). In certain embodiments, the sequencing information for the plurality of reporters provides information of mRNA expression levels for at least one reporter of the plurality of reporters. In certain embodiments, at least one reporter comprises a barcode sequence. In certain embodiments, the barcode sequence is operably linked to a promoter element and / or an enhancer element. In certain embodiments, the method further comprises performing a sequencing reaction on the sequencing library to obtain sequencing information for (i) the plurality of reporters, and (ii) the first plurality of control nucleic acids, the second plurality of control nucleic acids, and the fourth plurality of control nucleic acids. In certain embodiments, the first plurality of control nucleic acids comprises RNA nucleic acids.In certain embodiments, the first plurality of control nucleic acids is added to the plurality of cells before lysis. In certain embodiments, the first plurality of control nucleic acids comprises a first nucleotide sequence, a second nucleotide sequence, a third nucleotide sequence, and / or a fourth nucleotide sequence that are distinguishable from each other. In certain embodiments, the method further comprises normalizing sequence information for at least one reporter of the plurality of reporters to the first plurality of control nucleic acids. In certain embodiments, the second plurality of control nucleic acids comprises double-stranded DNA nucleic acids. In certain embodiments, the third plurality of control nucleic acids comprises RNA nucleic acids. In certain embodiments, the method further comprises determining the relative abundance of the second plurality of control nucleic acids in the reverse transcribed nucleic acid. In certain embodiments, the fourth plurality of control nucleic acids comprises single-stranded DNA nucleic acids. In certain embodiments, the method further comprises determining a control sequence that is not a sequence of the plurality of reporters, the first plurality of control nucleic acids, the second plurality of control nucleic acids, and the fourth plurality of control nucleic acids. In certain embodiments, the plurality of reporters comprises at least 10 reporters, at least 100 reporters, at least 1,000 reporters, at least 5,000 reporters, at least 10,000 reporters. In certain embodiments, each of the at least 10 reporters, at least 100 reporters, at least 1,000 reporters, at least 5,000 reporters, at least 10,000 reporters comprises a unique barcode sequence. In certain embodiments, each of the at least 10 reporters, at least 100 reporters, at least 1,000 reporters, at least 5,000 reporters, at least 10,000 reporters uniquely identifies a target molecule. In certain embodiments, the target molecule is a polypeptide expressed by a plurality of cells. In certain embodiments, the sequencing reaction is a high-throughput sequencing reaction. [Brief description of the drawings]

[0006] The novel features described herein are set forth with particularity in the appended claims. A better understanding of the features described herein and the advantages thereof will be obtained by reference to the following detailed description and the accompanying drawings that set forth illustrative examples in which the principles of the features described herein are utilized.

[0007] [Figure 1] 1 shows an exemplary next generation sequencing read output from an HTS assay. Wells with read counts below the dotted line were rejected. [Diagram 2] A plate map of rejected wells was generated using the quality control measures described herein and is shown. [Diagram 3] Shown are read counts of double stranded DNA control spike-in for samples before and after treatment with DNAse. [Figure 4] Shown are the read counts of single stranded DNA control spike-in of samples before and after treatment with reverse transcription. [Diagram 5] 1 shows the read counts for a variety of control nucleic acids after one or more steps have been performed. [Figure 6] 1 shows an exemplary process of adding various multiple nucleic acid spikes. [Figure 7] 1 shows read counts for a variety of nucleic acids. [Figure 8] Relevant ratios for analyzing process performance and reliability data for various assays and steps within assays are presented. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] In the following description, certain specific details are set forth to provide a thorough understanding of various embodiments. However, one of ordinary skill in the art will appreciate that the provided embodiments may be practiced without these details. Unless otherwise required by context, throughout this specification and the following claims, the word "comprise" and variations thereof (such as "comprises" and "comprising") shall be construed in an open and inclusive sense, i.e., "including, but not limited." As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. It should also be noted that the term "or" is generally used in the sense of including "and / or" unless the content clearly dictates otherwise. Additionally, the headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.

[0009] As used herein, the term "about" refers to an amount around 10% or less of the indicated amount.

[0010] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Target polypeptides provided, e.g., polypeptides including linkers and connecting peptides, may contain amino acid residues including natural amino acid residues and / or non-natural amino acid residues. The term also includes post-expression modifications of the polypeptide, e.g., glycosylation, sialylation, acetylation, phosphorylation, and the like. In some embodiments, a polypeptide may contain modifications to the native or natural sequence, so long as the protein maintains a desired activity. These modifications may be deliberate, such as by site-directed mutagenesis, or may be accidental, such as by mutation of the host that produces the protein or by imperfections due to PCR amplification.

[0011] Percent sequence identity (%) with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence after aligning the sequences and introducing gaps as necessary to obtain the maximum percent sequence identity, and any conservative substitutions are not considered as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved by various known means, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences can be determined, including the algorithm required to achieve maximum alignment over the entire length of the sequences being compared. However, for the purposes herein, % amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington, DC, 20559, and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, Calif.) or may be compiled from its source code. The ALIGN-2 program is compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0012] In the context of using ALIGN-2 for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be said to be a given amino acid sequence A having or containing a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: multiply the fraction X / Y by 100, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not comparable to the length of amino acid sequence B, the % amino acid sequence identity of A to B is not comparable to the % amino acid sequence identity of B to A. Unless specifically mentioned, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0013] The polypeptides described herein can be encoded by nucleic acids. A nucleic acid is a type of polynucleotide that contains two or more nucleotide bases. In certain embodiments, a nucleic acid is a component of a vector that can be used to transcribe a polynucleotide encoding a polypeptide into a cell. As used herein, the term "vector" refers to a nucleic acid molecule that can transport another nucleic acid to which it is linked. One type of vector is a genome-integrating vector or "integrating vector" that can be integrated into the chromosomal DNA of a host cell. Another type of vector is an "episomal" vector, e.g., a nucleic acid that can replicate extrachromosomally. A vector capable of directing the expression of a gene to which it is operably linked is referred to herein as an "expression vector." Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors, and the like. In expression vectors, regulatory elements such as promoters, enhancers, polyadenylation signals, and the like, used to control transcription can be derived from mammalian, bacterial, viral, or insect genes. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants can also be incorporated. Vectors derived from viruses, such as lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, etc., may be used. Plasmid vectors can be linearized for integration into chromosomal locations. Vectors may contain sequences that direct site-specific integration (e.g., AttP-AttB recombination) into a defined location or set of restricted sites within the genome. Additionally, vectors may contain sequences derived from transposable elements.

[0014] As used herein, the terms "homologous", "homology", or "percent homology" when used herein to describe a reference sequence, an amino acid sequence, or a nucleic acid sequence, may be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990, modified as in Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such formula is incorporated into the basic local alignment search tool (BLAST) program of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). Percent homology of sequences may be determined using the most recent version of BLAST as of the filing date of this application.

[0015] Assay Described herein is a method of quality control for high-throughput screening assays. Such assays are cell-based and include multiple cells expressing different potential target polypeptides. The activity of, for example, a screening library of small molecules or polypeptides against a target polypeptide can be determined using various reporter assays that can be activated by the target polypeptide or the activity of the target polypeptide by downstream mediators. In certain embodiments, the reporter comprises a barcode (e.g., an index sequence, also referred to herein as a "unique molecular identifier") that is uniquely mapped to the target polypeptide and allows activity on the polypeptide to be determined by a sequencing assay. In general, a library of cells containing different target polypeptides may comprise at least 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 or more different polypeptides that are uniquely labeled by barcodes. The assays of the present disclosure can be performed in 96-well plates, 384-well plates, or 1,536-well plates. The reporter may also generally include a visually traceable gene (e.g., a fluorescent or luminescent reporter gene) that can mark the activation of the reporter. In the assays described herein, different cells containing different target polypeptides (and barcodes) may be present in the same well. In certain embodiments, some or all of the cells may not express a particular target and may include a biological reporter that includes a UMI. Such a biological reporter may include a promoter, repressor, or enhancer element operably linked to the UMI, and optionally an additional reporter gene such as a luciferase enzyme or a fluorescent protein.

[0016] A non-limiting example of an assay design according to the methods described herein may include the steps of: 1) seeding cells in wells of a well plate; 2) contacting the cells with a test agent and allowing time for the test agent to affect reporter activation (e.g., expression of a barcode); 3) lysing the cells to release nucleic acids from the cells; 4) treating the lysate with DNAse to remove contaminating genomic DNA; 5) reverse transcribing and / or amplifying the reverse transcribed mRNA to generate cDNA or amplified cDNA (e.g., RT-PCR); 6) preparing the cDNA for next-generation sequencing reaction; and 7) sequencing the prepared cDNA. The above steps may be performed for each well of the assay. For steps 3)-7), various nucleic acids are released and / or processed after the cells are seeded in the wells and treated with the test agent. For the lysis step, nucleic acids are released from the cells. These nucleic acids may be treated with DNAse, which cleaves and removes double-stranded DNA. The RNA is then reverse transcribed into single-stranded DNA by reverse transcription, which is then further amplified by PCR to produce double-stranded DNA. Thus, multiple nucleic acids may be introduced and / or generated throughout the steps of the assay, and each of the nucleic acids may be sequenced and identified. The effectiveness of each step may be evaluated using the read count, amount, or ratio of control nucleic acid detected during each step, or in certain embodiments, using control nucleic acid sequences (also referred to herein as "spike-ins" of various nucleic acids when added to the wells of the assay during the process steps) in addition to the control nucleic acids added during the various steps, or in certain embodiments, at the final sequencing stage.

[0017] For each step in the above non-limiting examples, certain conditions may affect the effectiveness of each step (e.g., how many cells are lysed during the lysis step, the efficiency of the DNase step, how much cDNA or amplified cDNA is generated during the reverse transcription step, etc.), which may affect how many cDNA reads are associated with or converted to that step, which indicates which conditions lead to better performance or transfer when using assays with different conditions. As described below, the use of control nucleic acids that can be identified throughout the method allows for an indication of how efficient each step is, which can lead to optimization of the steps by modifying the conditions so that the step is as efficient as possible. These process controls can also increase the ability to normalize and compare data across assays or plates. These process controls can also identify sections or wells of the assay that should be excluded from the analysis.

[0018] Furthermore, the read counts of different control nucleic acids during different steps can indicate whether each step of the process was performed efficiently or not. For example, if 10,000 nucleic acids (e.g., DNA spike-in) are added to a well during the lysis step, and these 10,000 nucleic acids remain after the DNase step, which is intended to cleave the nucleic acids, and the read counts of these nucleic acids are 10,000, it can be determined that the DNase step is not efficient in that particular well, and therefore the data should be excluded. Conversely, if 10,000 RNA spike-ins are added during RT-PCR, where the RNA spike-ins are expected to eventually be converted to DNA, and the calculated DNA read count at the end of the RT-PCR step is 10,000 reads, even though the expected read count is 5,000 reads, it can be determined that at least one step of the assay or the measurement of the assay is either incorrect or inefficient, since the actual read count is much larger than the expected read count, and therefore the data from that well should be excluded.

[0019] Control Nucleic Acid Control nucleic acids may be added during one or more of the lysis step, DNase step, reverse transcription reaction, RT-PCR reaction, or next generation sequencing library preparation reaction. The control nucleic acids may contain known sequences that differ among different types of control nucleic acids to identify variability in different process steps.

[0020] FIG. 6 illustrates an exemplary process in which control nucleic acids are added during various steps, such as the lysis step, the DNase step, and the RT-PCR step, as described above. When added to the wells during the steps of the assay, the control nucleic acids may be referred to as "spike-ins" of various nucleic acids (e.g., DNA or RNA). Comparing the known amount of control nucleic acid to the amount of reads of the nucleic acid that is subsequently sequenced provides insight into the effect of each step. The amount of control nucleic acid added at each step may vary. Additionally, the amount of control nucleic acid may be limited within a range (e.g., 1,000-10,000 control nucleic acids among multiple nucleic acids) so as not to interfere with the effect of each step. For example, if too little control nucleic acid is added during a step, not enough control nucleic acid is added to calibrate the step (e.g., change one or more conditions of the step) or obtain a preferred read count (e.g., 1,000 reads per well). Conversely, adding too much control nucleic acid can interfere with the observable performance of the process (e.g., there may be too much nucleic acid spike-in, which uses too much of the reagents available to obtain a read from the reporter-generated UMI), thus affecting read counts.

[0021] Control nucleic acids may be added to the sample at various process steps based on absolute numbers (or concentrations equivalent to absolute numbers). In some embodiments, the amount of control nucleic acid (e.g., RNA or DNA spike-in) added at a particular process step comprises from about 1,000 nucleic acids to about 50,000 nucleic acids. In some embodiments, the amount of nucleic acid (e.g., RNA or DNA spike-in) added at a particular process step comprises from about 1,000 nucleic acids to about 2,000 nucleic acids, from about 1,000 nucleic acids to about 3,000 nucleic acids, from about 1,000 nucleic acids to about 4,000 nucleic acids, from about 1,000 nucleic acids to about 5,000 nucleic acids, from about 1,000 nucleic acids to about 7,500 nucleic acids, from about 1,000 nucleic acids to about 10,000 nucleic acids, from about 1,000 nucleic acids to about 12,500 nucleic acids, .... About 15,000 nucleic acids, about 1,000 nucleic acids to about 20,000 nucleic acids, about 1,000 nucleic acids to about 30,000 nucleic acids, about 1,000 nucleic acids to about 50,000 nucleic acids, about 2,000 nucleic acids to about 3,000 nucleic acids, about 2,000 nucleic acids to about 4,000 nucleic acids, about 2,000 nucleic acids to about 5,000 nucleic acids, about 2,000 nucleic acids to about 7,500 nucleic acids, about 2,000 nucleic acids to about 10,000 nucleic acids, about 2,000 nucleic acids to about 1 2,500 nucleic acids, about 2,000 nucleic acids to about 15,000 nucleic acids, about 2,000 nucleic acids to about 20,000 nucleic acids, about 2,000 nucleic acids to about 30,000 nucleic acids, about 2,000 nucleic acids to about 50,000 nucleic acids, about 3,000 nucleic acids to about 4,000 nucleic acids, about 3,000 nucleic acids to about 5,000 nucleic acids, about 3,000 nucleic acids to about 7,500 nucleic acids, about 3,000 nucleic acids to about 10,000 nucleic acids, about 3,000 nucleic acids to about 12 ,500 nucleic acids, about 3,000 nucleic acids to about 15,000 nucleic acids, about 3,000 nucleic acids to about 20,000 nucleic acids, about 3,000 nucleic acids to about 30,000 nucleic acids, about 3,000 nucleic acids to about 50,000 nucleic acids, about 4,000 nucleic acids to about 5,000 nucleic acids, about 4,000 nucleic acids to about 7,500 nucleic acids, about 4,000 nucleic acids to about 10,000 nucleic acids, about 4,000 nucleic acids to about 12,500 nucleic acids, about 4,000 nucleic acids to about 15,000 nucleic acids, about 4,000 nucleic acids to about 20,000 nucleic acids, about 4,000 nucleic acids to about 30,000 nucleic acids, about 4,000 nucleic acids to about 50,000 nucleic acids, about 5,000 nucleic acids to about 7,500 nucleic acids, about 5,000 nucleic acids to about 10,000 nucleic acids, about 5,000 nucleic acids to about 12,500 nucleic acids, about 5,000 nucleic acids to about 15,000 nucleic acids, about 5,000 nucleic acids to about 20,000 0 nucleic acids, about 5,000 nucleic acids to about 30,000 nucleic acids, about 5,000 nucleic acids to about 50,000 nucleic acids, about 7,500 nucleic acids to about 10,000 nucleic acids, about 7,500 nucleic acids to about 12,500 nucleic acids, about 7,500 nucleic acids to about 15,000 nucleic acids, about 7,500 nucleic acids to about 20,000 nucleic acids, about 7,500 nucleic acids to about 30,000 nucleic acids, about 7,500 nucleic acids to about 50,000 nucleic acids nucleic acids, about 10,000 nucleic acids to about 12,500 nucleic acids, about 10,000 nucleic acids to about 15,000 nucleic acids, about 10,000 nucleic acids to about 20,000 nucleic acids, about 10,000 nucleic acids to about 30,000 nucleic acids, about 10,000 nucleic acids to about 50,000 nucleic acids, about 12,500 nucleic acids to about 15,000 nucleic acids, about 12,500 nucleic acids to about 20,000 nucleic acids, about 12,500 nucleic acids to about About 30,000 nucleic acids, about 12,500 nucleic acids to about 50,000 nucleic acids, about 15,000 nucleic acids to about 20,000 nucleic acids, about 15,000 nucleic acids to about 30,000 nucleic acids, about 15,000 nucleic acids to about 50,000 nucleic acids, about 20,000 nucleic acids to about 30,000 nucleic acids, about 20,000 nucleic acids to about 50,000 nucleic acids, or about 30,000 nucleic acids to about 50,000 nucleic acids. In some embodiments, the amount of nucleic acids (e.g., RNA or DNA spike-in) is about 1,000 nucleic acids, about 2,000 nucleic acids, about 3,000 nucleic acids, about 4,000 nucleic acids, about 5,000 nucleic acids, about 7,500 nucleic acids, about 10,000 nucleic acids, about 12,500 nucleic acids, about 15,000 nucleic acids, about 20,000 nucleic acids, about 30,000 nucleic acids, or about 50,In some embodiments, the amount of nucleic acid (e.g., RNA or DNA spike-in) comprises at least about 1,000 nucleic acids, about 2,000 nucleic acids, about 3,000 nucleic acids, about 4,000 nucleic acids, about 5,000 nucleic acids, about 7,500 nucleic acids, about 10,000 nucleic acids, about 12,500 nucleic acids, about 15,000 nucleic acids, about 20,000 nucleic acids, or about 30,000 nucleic acids. In some embodiments, the amount of nucleic acid (e.g., RNA or DNA spike-in) comprises up to about 2,000 nucleic acids, about 3,000 nucleic acids, about 4,000 nucleic acids, about 5,000 nucleic acids, about 7,500 nucleic acids, about 10,000 nucleic acids, about 12,500 nucleic acids, about 15,000 nucleic acids, about 20,000 nucleic acids, about 30,000 nucleic acids, or about 50,000 nucleic acids. These spike-ins may comprise dsDNA, ssDNA, ssRNA, or combinations thereof. The amount of nucleic acid (e.g., read count / absolute number) may be used to determine the concentration of the nucleic acid in the well, and the concentration of the nucleic acid in the well may be used to determine the amount of the nucleic acid in the well. In some embodiments, a known amount of nucleic acid may be added to the well at the beginning of the method or process, and the concentration of the nucleic acid may be determined after the method or process is performed.

[0022] In some embodiments, the control nucleic acids added at various steps of the assay result in a certain total number of reads for all of the different control nucleic acids, and the expected read count per well in the assay is about 1,000 reads of the control nucleic acid. In some embodiments, the read count per well in the assay is about 0 to about 150 control nucleic acids. In some embodiments, the read count per well in the assay is about 0 to about 5 control nucleic acids, about 0 to about 10 control nucleic acids, about 0 to about 15 control nucleic acids, about 0 to about 20 control nucleic acids, about 0 to about 25 control nucleic acids, about 0 to about 30 control nucleic acids, about 0 to about 40 control nucleic acids, about 0 to about 50 ... About 75 control nucleic acids, about 0 control nucleic acids to about 100 control nucleic acids, about 0 control nucleic acids to about 150 control nucleic acids, about 5 control nucleic acids to about 10 control nucleic acids, about 5 control nucleic acids to about 15 control nucleic acids, about 5 control nucleic acids to about 20 control nucleic acids, about 5 control nucleic acids to about 25 control nucleic acids, about 5 control nucleic acids to about 30 control nucleic acids, about 5 control nucleic acids to about 40 control nucleic acids, about 5 control nucleic acids to about 50 control nucleic acids, about 5 control nucleic acids to about 75 control nucleic acids, about 5 control nucleic acids to about 100 control nucleic acids, about 5 control nucleic acids to about 150 control nucleic acids, about 10 control nucleic acids to about 15 control nucleic acids, about 10 control nucleic acids to about 20 control nucleic acids, about 10 control nucleic acids to about 25 control nucleic acids, about 10 control nucleic acids to about 30 control nucleic acids, about 10 control nucleic acids to about 40 control nucleic acids, about 10 control nucleic acids to about 50 control nucleic acids, about 10 control nucleic acids to about 75 control nucleic acids, about 10 control nucleic acids from about 100 control nucleic acids, from about 10 control nucleic acids to about 150 control nucleic acids, from about 15 control nucleic acids to about 20 control nucleic acids, from about 15 control nucleic acids to about 25 control nucleic acids, from about 15 control nucleic acids to about 30 control nucleic acids, from about 15 control nucleic acids to about 40 control nucleic acids, from about 15 control nucleic acids to about 50 control nucleic acids, from about 15 control nucleic acids to about 75 control nucleic acids, from about 15 control nucleic acids to about 100 control nucleic acids, from about 15 control nucleic acids to about 150 control nucleic acids,From about 20 control nucleic acids to about 25 control nucleic acids, from about 20 control nucleic acids to about 30 control nucleic acids, from about 20 control nucleic acids to about 40 control nucleic acids, from about 20 control nucleic acids to about 50 control nucleic acids, from about 20 control nucleic acids to about 75 control nucleic acids, from about 20 control nucleic acids to about 100 control nucleic acids, from about 20 control nucleic acids to about 150 control nucleic acids, from about 25 control nucleic acids to about 30 control nucleic acids, from about 25 control nucleic acids to about 40 control nucleic acids, from about 25 control nucleic acids to about 50 control nucleic acids, from about 25 control nucleic acids to about 75 control nucleic acids, from about 25 control nucleic acids to about 100 control nucleic acids, from about 25 control nucleic acids to about 150 control nucleic acids, from about 30 control nucleic acids to about 40 control nucleic acids, from about 30 control nucleic acids from about 50 control nucleic acids, from about 30 control nucleic acids to about 75 control nucleic acids, from about 30 control nucleic acids to about 100 control nucleic acids, from about 30 control nucleic acids to about 150 control nucleic acids, from about 40 control nucleic acids to about 50 control nucleic acids, from about 40 control nucleic acids to about 75 control nucleic acids, from about 40 control nucleic acids to about 100 control nucleic acids, from about 40 control nucleic acids to about 150 control nucleic acids, from about 50 control nucleic acids to about 75 control nucleic acids, from about 50 control nucleic acids to about 100 control nucleic acids, from about 50 control nucleic acids to about 150 control nucleic acids, from about 75 control nucleic acids to about 100 control nucleic acids, from about 75 control nucleic acids to about 150 control nucleic acids, or from about 100 control nucleic acids to about 150 control nucleic acids. In some embodiments, the read count per well in the assay is about 0 control nucleic acid, about 5 control nucleic acid, about 10 control nucleic acid, about 15 control nucleic acid, about 20 control nucleic acid, about 25 control nucleic acid, about 30 control nucleic acid, about 40 control nucleic acid, about 50 control nucleic acid, about 75 control nucleic acid, about 100 control nucleic acid, or about 150 control nucleic acid. In some embodiments, the read count per well in the assay is at least about 0 control nucleic acid, about 5 control nucleic acid, about 10 control nucleic acid, about 15 control nucleic acid, about 20 control nucleic acid, about 25 control nucleic acid, about 30 control nucleic acid, about 40 control nucleic acid, about 50 control nucleic acid, about 75 control nucleic acid, or about 100 control nucleic acid. In some embodiments, the read count per well in the assay is at most about 5 control nucleic acid, about 10 control nucleic acid, about 15 control nucleic acid,About 20 control nucleic acids, about 25 control nucleic acids, about 30 control nucleic acids, about 40 control nucleic acids, about 50 control nucleic acids, about 75 control nucleic acids, about 100 control nucleic acids, or about 150 control nucleic acids. In some embodiments, the read count per well in the assay is about 100 control nucleic acids to about 1,500 control nucleic acids. In some embodiments, the read count per well in the assay is about 100 control nucleic acids to about 200 control nucleic acids, about 100 control nucleic acids to about 300 control nucleic acids, about 100 control nucleic acids to about 400 control nucleic acids, about 100 control nucleic acids to about 500 control nucleic acids, about 100 control nucleic acids to about 600 control nucleic acids, about 100 control nucleic acids to about 700 control nucleic acids, about 100 control nucleic acids to about 800 control nucleic acids, about 100 control nucleic acids to about 1,500 control nucleic acids. About 900 control nucleic acids, about 100 control nucleic acids to about 1,000 control nucleic acids, about 100 control nucleic acids to about 1,250 control nucleic acids, about 100 control nucleic acids to about 1,500 control nucleic acids, about 200 control nucleic acids to about 300 control nucleic acids, about 200 control nucleic acids to about 400 control nucleic acids, about 200 control nucleic acids to about 500 control nucleic acids, about 200 control nucleic acids to about 600 control nucleic acids, about 200 control nucleic acids to about 700 control nucleic acids, about 200 control nucleic acids From about 800 control nucleic acids, from about 200 control nucleic acids to about 900 control nucleic acids, from about 200 control nucleic acids to about 1,000 control nucleic acids, from about 200 control nucleic acids to about 1,250 control nucleic acids, from about 200 control nucleic acids to about 1,500 control nucleic acids, from about 300 control nucleic acids to about 400 control nucleic acids, from about 300 control nucleic acids to about 500 control nucleic acids, from about 300 control nucleic acids to about 600 control nucleic acids, from about 300 control nucleic acids to about 700 control nucleic acids, from about 300 control nucleic acids to about 800 control nucleic acids, about 300 control nucleic acids to about 900 control nucleic acids, about 300 control nucleic acids to about 1,000 control nucleic acids, about 300 control nucleic acids to about 1,250 control nucleic acids, about 300 control nucleic acids to about 1,500 control nucleic acids, about 400 control nucleic acids to about 500 control nucleic acids, about 400 control nucleic acids to about 600 control nucleic acids, about 400 control nucleic acids to about 700 control nucleic acids, about 400 control nucleic acids to about 800 control nucleic acids,About 400 control nucleic acids to about 900 control nucleic acids, about 400 control nucleic acids to about 1,000 control nucleic acids, about 400 control nucleic acids to about 1,250 control nucleic acids, about 400 control nucleic acids to about 1,500 control nucleic acids, about 500 control nucleic acids to about 600 control nucleic acids, about 500 control nucleic acids to about 700 control nucleic acids, about 500 control nucleic acids to about 800 control nucleic acids, about 500 control nucleic acids to about 900 control nucleic acids, about 500 control nucleic acids From about 1,000 control nucleic acids, from about 500 control nucleic acids to about 1,250 control nucleic acids, from about 500 control nucleic acids to about 1,500 control nucleic acids, from about 600 control nucleic acids to about 700 control nucleic acids, from about 600 control nucleic acids to about 800 control nucleic acids, from about 600 control nucleic acids to about 900 control nucleic acids, from about 600 control nucleic acids to about 1,000 control nucleic acids, from about 600 control nucleic acids to about 1,250 control nucleic acids, from about 6 ... from about 1,500 control nucleic acids, from about 700 control nucleic acids to about 800 control nucleic acids, from about 700 control nucleic acids to about 900 control nucleic acids, from about 700 control nucleic acids to about 1,000 control nucleic acids, from about 700 control nucleic acids to about 1,250 control nucleic acids, from about 700 control nucleic acids to about 1,500 control nucleic acids, from about 800 control nucleic acids to about 900 control nucleic acids, from about 800 control nucleic acids to about 1,000 control nucleic acids, from about 800 control nucleic acids to about 1 ,250 control nucleic acids, about 800 control nucleic acids to about 1,500 control nucleic acids, about 900 control nucleic acids to about 1,000 control nucleic acids, about 900 control nucleic acids to about 1,250 control nucleic acids, about 900 control nucleic acids to about 1,500 control nucleic acids, about 1,000 control nucleic acids to about 1,250 control nucleic acids, about 1,000 control nucleic acids to about 1,500 control nucleic acids, or about 1,250 control nucleic acids to about 1,500 control nucleic acids. In some embodiments, the read count per well in the assay is about 100 control nucleic acids, about 200 control nucleic acids, about 300 control nucleic acids, about 400 control nucleic acids, about 500 control nucleic acids, about 600 control nucleic acids, about 700 control nucleic acids, about 800 control nucleic acids, about 900 control nucleic acids, about 1,000 control nucleic acids, about 1,250 control nucleic acids, or about 1,500 control nucleic acids.The read count per well in the assay is at least about 100 control nucleic acids, about 200 control nucleic acids, about 300 control nucleic acids, about 400 control nucleic acids, about 500 control nucleic acids, about 600 control nucleic acids, about 700 control nucleic acids, about 800 control nucleic acids, about 900 control nucleic acids, about 1,000 control nucleic acids, or about 1,250 control nucleic acids. In some embodiments, the read count per well in the assay is at most about 200 control nucleic acids, about 300 control nucleic acids, about 400 control nucleic acids, about 500 control nucleic acids, about 600 control nucleic acids, about 700 control nucleic acids, about 800 control nucleic acids, about 900 control nucleic acids, about 1,000 control nucleic acids, about 1,250 control nucleic acids, or about 1,500 control nucleic acids. In some embodiments, the read count per well in the assay is from about 500 control nucleic acids to about 15,000 control nucleic acids. In some embodiments, the read count per well in the assay is from about 500 control nucleic acids to about 750 control nucleic acids, from about 500 control nucleic acids to about 1,000 control nucleic acids, from about 500 control nucleic acids to about 1,250 control nucleic acids, from about 500 control nucleic acids to about 1,500 control nucleic acids, from about 500 control nucleic acids to about 2,000 control nucleic acids, from about 500 control nucleic acids to about 3,000 control nucleic acids, from about 500 control nucleic acids to about 4,000 control nucleic acids, from about 500 control nucleic acids to about 5,000 control nucleic acids, from about 500 control nucleic acids to about 7,500 control nucleic acids, from about 500 control nucleic acids to about 10,000 control nucleic acids, from about 500 control nucleic acids from about 15,000 control nucleic acids, from about 750 control nucleic acids to about 1,000 control nucleic acids, from about 750 control nucleic acids to about 1,250 control nucleic acids, from about 750 control nucleic acids to about 1,500 control nucleic acids, from about 750 control nucleic acids to about 2,000 control nucleic acids, from about 750 control nucleic acids to about 3,000 control nucleic acids, from about 750 control nucleic acids to about 4,000 control nucleic acids, from about 750 control nucleic acids to about 5,000 control nucleic acids, from about 750 control nucleic acids to about 7,500 control nucleic acids, from about 750 control nucleic acids to about 10,000 control nucleic acids, from about 750 control nucleic acids to about 15,000 control nucleic acids, from about 1,000 control nucleic acids to about 1,250 control nucleic acids,From about 1,000 control nucleic acids to about 1,500 control nucleic acids, from about 1,000 control nucleic acids to about 2,000 control nucleic acids, from about 1,000 control nucleic acids to about 3,000 control nucleic acids, from about 1,000 control nucleic acids to about 4,000 control nucleic acids, from about 1,000 control nucleic acids to about 5,000 control nucleic acids, from about 1,000 control nucleic acids to about 7,500 control nucleic acids, from about 1,000, From about 10,000 control nucleic acids, from about 1,000 control nucleic acids to about 15,000 control nucleic acids, from about 1,250 control nucleic acids to about 1,500 control nucleic acids, from about 1,250 control nucleic acids to about 2,000 control nucleic acids, from about 1,250 control nucleic acids to about 3,000 control nucleic acids, from about 1,250 control nucleic acids to about 4,000 control nucleic acids, from about 1,250 control nucleic acids to about 5,000 control nucleic acids, from about 1,250 control nucleic acids to about 7,500 control nucleic acids, from about 1,250 control nucleic acids to about 10,000 control nucleic acids, from about 1,250 control nucleic acids from about 15,000 control nucleic acids, from about 1,500 control nucleic acids to about 2,000 control nucleic acids, from about 1,500 control nucleic acids to about 3,000 control nucleic acids, from about 1,500 control nucleic acids to about 4,000 control nucleic acids, from about 1,500 control nucleic acids to about 5,000 control nucleic acids, from about 1,500 control nucleic acids to about 7,500 control nucleic acids, from about 1,500 control nucleic acids to about 10,000 control nucleic acids, from about 1,500 control nucleic acids to about 15,000 control nucleic acids, from about 2,000 control nucleic acids to about 3,000 control nucleic acids, from about 2,000 control nucleic acids About 4,000 control nucleic acids, about 2,000 control nucleic acids to about 5,000 control nucleic acids, about 2,000 control nucleic acids to about 7,500 control nucleic acids, about 2,000 control nucleic acids to about 10,000 control nucleic acids, about 2,000 control nucleic acids to about 15,000 control nucleic acids, about 3,000 control nucleic acids to about 4,000 control nucleic acids, about 3,000 control nucleic acids to about 5,000 control nucleic acids, about 3,000 control nucleic acids to about 7,500 control nucleic acids, about 3,000 control nucleic acids to about 10,000 control nucleic acids, about 3,000 control nucleic acids to about 15 ,000 control nucleic acids, from about 4,000 control nucleic acids to about 5,000 control nucleic acids, from about 4,000 control nucleic acids to about 7,500 control nucleic acids, from about 4,000 control nucleic acids to about 10,000 control nucleic acids, from about 4,000 control nucleic acids to about 15,000 control nucleic acids, from about 5,000 control nucleic acids to about 7,500 control nucleic acids, from about 5,000 control nucleic acids to about 10,000 control nucleic acids, from about 5,000 control nucleic acids to about 15,000 control nucleic acids, from about 7,500 control nucleic acids to about 10,000 control nucleic acids, from about 7,500 control nucleic acids to about 15,000 control nucleic acids, or about 10,000 to about 15,000 control nucleic acids. In some embodiments, the read counts per well in the assay are about 500 control nucleic acids, about 750 control nucleic acids, about 1,000 control nucleic acids, about 1,250 control nucleic acids, about 1,500 control nucleic acids, about 2,000 control nucleic acids, about 3,000 control nucleic acids, about 4,000 control nucleic acids, about 5,000 control nucleic acids, about 7,500 control nucleic acids, about 10,000 control nucleic acids, or about 15,000 control nucleic acids. In some embodiments, the read count per well in the assay is at least about 500 control nucleic acids, about 750 control nucleic acids, about 1,000 control nucleic acids, about 1,250 control nucleic acids, about 1,500 control nucleic acids, about 2,000 control nucleic acids, about 3,000 control nucleic acids, about 4,000 control nucleic acids, about 5,000 control nucleic acids, about 7,500 control nucleic acids, or about 10,000 control nucleic acids. In some embodiments, the read count per well in the assay is up to about 750 control nucleic acids, about 1,000 control nucleic acids, about 1,250 control nucleic acids, about 1,500 control nucleic acids, about 2,000 control nucleic acids, about 3,000 control nucleic acids, about 4,000 control nucleic acids, about 5,000 control nucleic acids, about 7,500 control nucleic acids, about 10,000 control nucleic acids, or about 15,000 control nucleic acids. Although specific read counts are described above, the read counts are exemplary and other read counts may be determined. Additionally, different read counts may be determined in wells for different control nucleic acids. Additionally, the same read count need not be determined between wells for all nucleic acids.

[0023] Adding a control nucleic acid (or a plurality of control nucleic acids) provides a method to determine the effectiveness of one or more steps. For example, a control nucleic acid may be introduced in a known amount during a step, and the reads of these control nucleic acids may be calculated later during the step in which the control nucleic acid is introduced, or may be calculated during a later step. Thus, the read count of the control nucleic acid during a particular step compared to the amount of control nucleic acid introduced may provide insight into how well the step is working (e.g., how efficient the reverse transcription reaction is). If the step is determined to be working efficiently (e.g., the reads of the control nucleic acid are near the expected amount of reads), the conditions of the step may not be changed, or may be changed to further improve the effectiveness of the step, but if the step is determined to be not working properly (e.g., the reads of the control nucleic acid are not near the expected amount of reads), the conditions of the step may be changed to improve the effectiveness.

[0024] The lysis step may include disrupting the cell membrane so that nucleic acids are released from inside the cell. The nucleic acids may include DNA, RNA, or other nucleic acids.

[0025] A first plurality of control nucleic acids (e.g., RNA spike 602) may be added during or before the lysis step. In certain embodiments, the first plurality of control nucleic acids may be included in a lysis agent or buffer that is added to the cells during cell lysis. In some embodiments, the first plurality of nucleic acids is present in a plurality of cells seeded in a well, and then the cells are lysed, releasing the internal contents of the cells. In certain embodiments, the nucleic acids include RNA. In certain embodiments, the nucleic acids include DNA. In certain embodiments, one of the first plurality of control nucleic acids is DNA and the other of the first plurality of control nucleic acids is RNA. This first plurality of nucleic acids may be used to (1) normalize end read counts to a known reference standard and / or (2) determine the ratio of read counts of the sample of interest versus the other control nucleic acid spike-in, which may be used as a quantitative measurement of the effectiveness of the cell lysis reaction. The first plurality of nucleic acids may be used to normalize end read counts to a known reference standard by comparing the read counts of the first plurality of nucleic acids during one or more subsequent steps (e.g., a sequencing step) with the amount of the first plurality of nucleic acids initially included. The first plurality of nucleic acids may also be used to determine a lysis reaction ratio of read counts by comparing the read counts of the first plurality of nucleic acids calculated during the sequencing step with the number of the first plurality of nucleic acids added to the lysis step. If the lysis reaction is inefficient, the lysis reaction ratio will be low, and if the lysis reaction is efficient, the lysis reaction ratio will be high. For example, a lysis reaction ratio of 0.1 indicates that the lysis reaction was inefficient because only 10% of the first plurality of nucleic acids were lysed. In contrast, a lysis reaction ratio of 0.95 indicates that the lysis reaction was efficient because 95% of the first plurality of nucleic acids were lysed. To improve the lysis reaction ratio, the conditions of the lysis step may be changed, such as the pH of the assay, the duration of the assay, the amount of cells added, or other conditions.

[0026] Other ratios, such as a lytic RNA stability ratio (e.g., "lytic RNA stability" 618), may be determined based on the additional nucleic acids added during the lysis step. For example, the lytic RNA stability ratio may measure the effect of transfer from the lysis step to the DNase step and / or determine whether the presence of RNase affects any of the subsequent steps before the RT step, including the RT step. Additionally, the lytic RNA stability ratio may indicate whether the presence of DNase in the well is too strong. For example, if there is too much DNase during the DNase step, the DNase may not only cleave DNA, but also RNA, which will result in a lower concentration of RNA, resulting in a lower lytic RNA stability ratio. Thus, the lytic RNA stability ratio may indicate that too much DNase is used during the DNase step. For example, if a read count of additional nucleic acids (e.g., additional RNA spikes) is performed at the end of the lysis step, and then another read count of additional nucleic acids is performed at the beginning of the DNase step, the effect of transfer may be evaluated using the lytic RNA stability ratio. Thus, a lytic RNA stability ratio of 0.94 indicates that the transfer was efficient. In contrast, a soluble RNA stability ratio of 0.25 indicates that transfer was inefficient and that certain conditions of transfer (e.g., rate, duration, temperature, and / or pH) may need to be altered.

[0027] A second plurality of control nucleic acids (e.g., DNA spike 604) may be added during or before the lysis step. In some embodiments, the second plurality of nucleic acids is among a plurality of cells added during the lysis step, which are then lysed. In certain embodiments, the second plurality of control nucleic acids may be included in a lysis agent or buffer that is added to the cells during cell lysis. In certain embodiments, these nucleic acids include double-stranded DNA. These double-stranded DNAs can be used to determine the effectiveness of the DNase step, and thus identify potential sources of contamination from incompletely DNase-treated samples. For example, the read counts of the second plurality of nucleic acids can indicate DNase inefficiency (e.g., "DNase inefficiency" 616). DNase inefficiency may be determined by comparing the read counts of RNA associated with the second plurality of nucleic acids measured during the DNase step to the read counts of RNA associated with the second plurality of nucleic acids measured during the reverse transcription step. In particular, all or a portion of the second plurality of nucleic acids added during the lysis step may be cleaved during the DNase step, and the resulting RNA is calculated during the DNase step. Thus, DNase inefficiency can be determined by comparing the read counts of RNA during the DNase step with the known amount of the second plurality of nucleic acids added during the lysis step. For example, a DNase inefficiency of 1,000:10,000 indicates that 90% of the second plurality of nucleic acids were cleaved during the DNase step. In contrast, a DNase inefficiency of 5,000:10,000 indicates that only 50% of the second plurality of nucleic acids were cleaved during the DNase step. To improve DNase inefficiency, the conditions of the lysis step may be altered, such as the pH of the assay, the duration of the assay, the amount of cells added, or other conditions.

[0028] A third plurality of nucleic acids (e.g., RNA spikes 608) may be added during the RT-PCR step. In certain embodiments, these nucleic acids include RNA. This plurality of nucleic acids may be used to identify insufficient or suboptimal nucleic acid transfer from the RT-PCR step to the sequencing step, any non-specific RNA digestion during the RT-PCR step, or the presence of contaminating RNases during the RT reaction. The RT-PCR RNA stability ratio may be determined based on how much RNA is transferred. For example, the third plurality of nucleic acids (e.g., RNA spikes) may be added in known amounts to a first location during the RT-PCR step and then transferred to a second location for the sequencing step to take place. If the transfer was efficient, the RT-PCR RNA stability ratio will be high, but if the transfer was inefficient, the RT-PCR RNA stability ratio will be low. For example, an RT-PCR RNA stability ratio of 0.3 indicates that only 30% of the RNA spikes were transferred, and therefore the transfer from the RT-PCR step to the sequencing step was inefficient. In contrast, an RT-PCR RNA stability ratio of 0.99 indicates that 99% of the RNA spike has been transferred and therefore the transfer from the RT-PCR step to the sequencing step is valid. If the RT-PCR RNA stability ratio is low, certain conditions associated with the step may be altered, such as the rate of transfer or the amount of RNA spike.

[0029] A fourth plurality of nucleic acids (e.g., DNA spikes 610) may be added during the RT-PCR process. In certain embodiments, these nucleic acids include single-stranded DNA (ssDNA). This spike-in can be used to determine the effectiveness of the reverse transcription portion of the RT-PCR process. The fourth plurality of nucleic acids may include single-stranded DNA spikes added during the reverse transcription step, and the read counts of the fourth plurality of nucleic acids at various steps can provide insight into how much complementary DNA is generated, thereby indicating the efficiency of the RT-PCR performance and the RT efficiency (e.g., "RT efficiency" 612). For example, a known amount of the fourth plurality of nucleic acids (e.g., ssDNA spikes) may be added during the RT-PCR process, and the known amount may be compared to the read counts of the fourth plurality of nucleic acids during or after the RT-PCR process. For example, an RT efficiency of 0.8 indicates that 80% of the ssDNA is converted by the RT-PCR process. In contrast, an RT efficiency of 0.1 indicates that only 10% of the ssDNA is converted by the RT-PCR process. Low RT efficiency may lead to alterations in the conditions of the RT-PCR step, for example, the temperature of the RT-PCR step, the pH of the RT-PCR step, and / or the duration of the RT-PCR step.

[0030] A fifth plurality of nucleic acids (e.g., RNA spikes 606) may be added during the DNase step. In certain embodiments, these nucleic acids include RNA spikes added at the DNase step. This spike-in can be used to determine the effectiveness of transfer from the DNase plate to the RT-PCR plate. DNase RNA stability (e.g., "DNase RNA Stability" 614) may be determined based on how much RNA is transferred. For example, the fifth plurality of nucleic acids (e.g., RNA spikes) may be added in known amounts to a first location (e.g., DNase plate) during the DNase step and then transferred to a second location (e.g., RT-PCR plate) for the reverse transcription step to occur. If the transfer was efficient, the DNase RNA stability ratio will be high, but if the transfer was inefficient, the DNase RNA stability ratio will be low. For example, a DNase RNA stability ratio of 0.3 indicates that only 30% of the RNA spikes were transferred, and therefore the transfer from the DNase step to the reverse transcription step was inefficient. In contrast, a DNase RNA Stability Ratio of 0.99 indicates that 99% of the RNA spike has been transferred and thus the transfer from the DNase step to the reverse transcription step is ineffective. If the DNase RNA Stability Ratio is low, certain conditions associated with the step may be altered, such as the rate of transfer or the amount of RNA spike.

[0031] Further information may indicate further aspects related to the process, such as further ratios determined based on the plurality of nucleic acids. For example, determining a gDNA contamination ratio (e.g., "gDNA Contamination" 620) indicates gDNA contamination occurring after adding a first plurality of nucleic acids. For example, a first plurality of nucleic acids (e.g., double-stranded DNA spikes) may be added in known amounts during the lysis step, which may be compared to the read counts of the first plurality of nucleic acids during the RT-PCR step to determine what percentage of DNA is gDNA. Thus, a gDNA contamination ratio of 0.75 indicates that the DNase step converted most of the DNA spike-in added during the lysis step into RNA. In contrast, a gDNA contamination ratio of 0.25 indicates that the DNase step converted most of the DNA spike-in added during the lysis step into RNA.

[0032] The nucleotide sequence (e.g., barcode) of the control nucleic acid described herein can be changed for a subsequent experimental run to detect cross-contamination from a previous experimental run. Such cross-contamination may be due to equipment or reagents used in a previous experimental run. In some embodiments, the plurality of nucleic acids may have commonly sequenced nucleotides. In other embodiments, each of the plurality of nucleic acids has its own nucleotide sequence.

[0033] Furthermore, as described above, various multiple nucleic acids may be added during one or more steps. In some embodiments, a single multiple nucleic acid may be added and evaluated throughout each step. For example, a single multiple nucleic acid (e.g., RNA spike) may be added during the lysis step, calculated at the end of the lysis step, calculated at the beginning of the DNase step, calculated at the end of the DNase step, calculated at the beginning of the RT-PCR step, calculated at the end of the RT-PCR step, and then calculated again during the sequencing step, effectively allowing multiple read counts of the single multiple nucleic acid to determine some of the ratios specified above, such as lysis RNA stability ratio, DNase RNA stability ratio, and RT-PCR RNA stability ratio.

[0034] In some embodiments, the nucleic acids may be present in a solution at a concentration. In some embodiments, a solvent may be added to achieve the concentration in the solution. In some embodiments, the concentration may be from about 0 pM to about 0.05 pM. In some embodiments, the concentration may be from about 0 pM to about 0.01 pM, from about 0 pM to about 0.015 pM, from about 0 pM to about 0.02 pM, from about 0 pM to about 0.025 pM, from about 0 pM to about 0.03 pM, from about 0 pM to about 0.035 pM, from about 0 pM to about 0.04 pM, from about 0 pM to about 0.045 pM, from about 0 pM to about 0.05 pM, from about 0.01 pM to about 0.015 pM, from about 0.01 pM to about 0.02 pM, from about 0.01 pM to about 0.025 pM , about 0.01 pM to about 0.03 pM, about 0.01 pM to about 0.035 pM, about 0.01 pM to about 0.04 pM, about 0.01 pM to about 0.045 pM, about 0.01 pM to about 0.05 pM, about 0.015 pM to about 0.02 pM, about 0.015 pM to about 0.025 pM, about 0.015 pM to about 0.03 pM, about 0.015 pM to about 0.035 pM, about 0.015 pM to about 0.04 pM, about 0.015 pM to about 0.045 pM, about 0 .015pM to about 0.05pM, about 0.02pM to about 0.025pM, about 0.02pM to about 0.03pM, about 0.02pM to about 0.035pM, about 0.02pM to about 0.04pM, about 0.02pM to about 0.045pM, about 0.02pM to about 0.05pM, about 0.025pM to about 0.03pM, about 0.025pM to about 0.035pM, about 0.025pM to about 0.04pM, about 0.025pM to about 0.045pM, about 0.02 It may be from 5 pM to about 0.05 pM, from about 0.03 pM to about 0.035 pM, from about 0.03 pM to about 0.04 pM, from about 0.03 pM to about 0.045 pM, from about 0.03 pM to about 0.05 pM, from about 0.035 pM to about 0.04 pM, from about 0.035 pM to about 0.045 pM, from about 0.035 pM to about 0.05 pM, from about 0.04 pM to about 0.045 pM, from about 0.04 pM to about 0.05 pM, or from about 0.045 pM to about 0.05 pM.In some embodiments, the concentration can be about 0 pM, about 0.01 pM, about 0.015 pM, about 0.02 pM, about 0.025 pM, about 0.03 pM, about 0.035 pM, about 0.04 pM, about 0.045 pM, or about 0.05 pM. In some embodiments, the concentration can be at least about 0 pM, about 0.01 pM, about 0.015 pM, about 0.02 pM, about 0.025 pM, about 0.03 pM, about 0.035 pM, about 0.04 pM, or about 0.045 pM. In some embodiments, the concentration can be up to about 0.01 pM, about 0.015 pM, about 0.02 pM, about 0.025 pM, about 0.03 pM, about 0.035 pM, about 0.04 pM, about 0.045 pM, or about 0.05 pM. In some embodiments, the concentration can be from about 0.05 pM to about 0.5 pM.In some embodiments, the concentration is from about 0.05 pM to about 0.1 pM, from about 0.05 pM to about 0.15 pM, from about 0.05 pM to about 0.2 pM, from about 0.05 pM to about 0.25 pM, from about 0.05 pM to about 0.3 pM, from about 0.05 pM to about 0.35 pM, from about 0.05 pM to about 0.4 pM, from about 0.05 pM to about 0.45 pM, from about 0.05 pM to about 0.5 pM, from about 0.1 pM to about 0.15 pM, from about 0.1 pM to about 0.2 pM, about 0.1 pM to about 0.25 pM, about 0.1 pM to about 0.3 pM, about 0.1 pM to about 0.35 pM, about 0.1 pM to about 0.4 pM, about 0.1 pM to about 0.45 pM, about 0.1 pM to about 0.5 pM, about 0.15 pM to about 0.2 pM, about 0.15 pM to about 0.25 pM, about 0.15 pM to about 0.3 pM, about 0.15 pM to about 0.35 pM, about 0.15 pM to about 0.4 pM, about 0.15 pM to about 0.45 pM, about 0.15 pM to about 0.5 pM, about 0.2 pM to about 0.25 pM, about 0.2 pM to about 0.3 pM, about 0.2 pM to about 0.35 pM, about 0.2 pM to about 0.4 pM, about 0.2 pM to about 0.45 pM, about 0.2 pM to about 0.5 pM, about 0.25 pM to about 0.3 pM, about 0.25 pM to about 0.35 pM, about 0.25 pM to about 0.4 pM, about 0.25 pM to about 0. It can be about 45 pM, about 0.25 pM to about 0.5 pM, about 0.3 pM to about 0.35 pM, about 0.3 pM to about 0.4 pM, about 0.3 pM to about 0.45 pM, about 0.3 pM to about 0.5 pM, about 0.35 pM to about 0.4 pM, about 0.35 pM to about 0.45 pM, about 0.35 pM to about 0.5 pM, about 0.4 pM to about 0.45 pM, about 0.4 pM to about 0.5 pM, or about 0.45 pM to about 0.5 pM. In some embodiments, the concentration can be about 0.05 pM, about 0.1 pM, about 0.15 pM, about 0.2 pM, about 0.25 pM, about 0.3 pM, about 0.35 pM, about 0.4 pM, about 0.45 pM, or about 0.5 pM. In some embodiments, the concentration can be at least about 0.05 pM, about 0.1 pM, about 0.15 pM, about 0.2 pM, about 0.25 pM, about 0.3 pM, about 0.35 pM, about 0.4 pM, or about 0.45 pM.In some embodiments, the concentration may be up to about 0.1 pM, about 0.15 pM, about 0.2 pM, about 0.25 pM, about 0.3 pM, about 0.35 pM, about 0.4 pM, about 0.45 pM, or about 0.5 pM. In some embodiments, the concentration may be from about 0.5 pM to about 2 pM. In some embodiments, the concentration may be from about 0.5 pM to about 0.6 pM, about 0.5 pM to about 0.7 pM, about 0.5 pM to about 0.8 pM, about 0.5 pM to about 0.9 pM, about 0.5 pM to about 1 pM, about 0.5 pM to about 1.25 pM, about 0.5 pM to about 1.5 pM, about 0.5 pM to about 1.75 pM, about 0.5 pM to about 2 pM, about 0.6 pM to about 0.7 pM, about 0.6 pM to about 0.6 ... pM to about 0.8 pM, about 0.6 pM to about 0.9 pM, about 0.6 pM to about 1 pM, about 0.6 pM to about 1.25 pM, about 0.6 pM to about 1.5 pM, about 0.6 pM to about 1.75 pM, about 0.6 pM to about 2 pM, about 0.7 pM to about 0.8 pM, about 0.7 pM to about 0.9 pM, about 0.7 pM to about 1 pM, about 0.7 pM to about 1.25 pM, about 0.7 pM to about 1.5 pM M, about 0.7 pM to about 1.75 pM, about 0.7 pM to about 2 pM, about 0.8 pM to about 0.9 pM, about 0.8 pM to about 1 pM, about 0.8 pM to about 1.25 pM, about 0.8 pM to about 1.5 pM, about 0.8 pM to about 1.75 pM, about 0.8 pM to about 2 pM, about 0.9 pM to about 1 pM, about 0.9 pM to about 1.25 pM, about 0.9 pM to about 1.5 pM, about 0.9 pM to about 1. It can be about 1.75 pM, about 0.9 pM to about 2 pM, about 1 pM to about 1.25 pM, about 1 pM to about 1.5 pM, about 1 pM to about 1.75 pM, about 1 pM to about 2 pM, about 1.25 pM to about 1.5 pM, about 1.25 pM to about 1.75 pM, about 1.25 pM to about 2 pM, about 1.5 pM to about 1.75 pM, about 1.5 pM to about 2 pM, or about 1.75 pM to about 2 pM. In some embodiments, the concentration can be about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.9 pM, about 1 pM, about 1.25 pM, about 1.5 pM, about 1.75 pM, or about 2 pM.In some embodiments, the concentration may be at least about 0.5 pM, about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.9 pM, about 1 pM, about 1.25 pM, about 1.5 pM, or about 1.75 pM. In some embodiments, the concentration may be up to about 0.6 pM, about 0.7 pM, about 0.8 pM, about 0.9 pM, about 1 pM, about 1.25 pM, about 1.5 pM, about 1.75 pM, or about 2 pM. In some embodiments, the concentration may be from about 1 pM to about 50 pM.In some embodiments, the concentrations are from about 1 pM to about 2 pM, from about 1 pM to about 3 pM, from about 1 pM to about 4 pM, from about 1 pM to about 5 pM, from about 1 pM to about 7.5 pM, from about 1 pM to about 10 pM, from about 1 pM to about 12.5 pM, from about 1 pM to about 15 pM, from about 1 pM to about 20 pM, from about 1 pM to about 25 pM, from about 1 pM to about 50 pM, from about 2 pM to about 3 pM, from about 2 pM to about 4 pM, from about 2 pM to about 5 pM, from about 2 pM to about 7.5 pM, from about 2 pM to about 10 pM, from about 2 pM to about 1 2.5pM, about 2pM to about 15pM, about 2pM to about 20pM, about 2pM to about 25pM, about 2pM to about 50pM, about 3pM to about 4pM, about 3pM to about 5pM, about 3pM to about 7.5pM, about 3pM to about 10pM, about 3pM to about 12.5pM, about 3pM to about 15pM, about 3pM to about 20pM, about 3pM to about 25pM, about 3pM to about 50pM, about 4pM to about 5pM, about 4pM to about 7.5pM, about 4pM to about 10pM, about 4pM to about 12.5pM, about 4p M to about 15 pM, about 4 pM to about 20 pM, about 4 pM to about 25 pM, about 4 pM to about 50 pM, about 5 pM to about 7.5 pM, about 5 pM to about 10 pM, about 5 pM to about 12.5 pM, about 5 pM to about 15 pM, about 5 pM to about 20 pM, about 5 pM to about 25 pM, about 5 pM to about 50 pM, about 7.5 pM to about 10 pM, about 7.5 pM to about 12.5 pM, about 7.5 pM to about 15 pM, about 7.5 pM to about 20 pM, about 7.5 pM to about 25 pM, about 7.5 pM to about 50 pM, about 10 pM to about 12.5 pM, about 10 pM to about 15 pM, about 10 pM to about 20 pM, about 10 pM to about 25 pM, about 10 pM to about 50 pM, about 12.5 pM to about 15 pM, about 12.5 pM to about 20 pM, about 12.5 pM to about 25 pM, about 12.5 pM to about 50 pM, about 15 pM to about 20 pM, about 15 pM to about 25 pM, about 15 pM to about 50 pM, about 20 pM to about 25 pM, about 20 pM to about 50 pM, or about 25 pM to about 50 pM. In some embodiments, the concentration can be about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 7.5 pM, about 10 pM, about 12.5 pM, about 15 pM, about 20 pM, about 25 pM, or about 50 pM.In some embodiments, the concentration may be at least about 1 pM, about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 7.5 pM, about 10 pM, about 12.5 pM, about 15 pM, about 20 pM, or about 25 pM. In some embodiments, the concentration may be up to about 2 pM, about 3 pM, about 4 pM, about 5 pM, about 7.5 pM, about 10 pM, about 12.5 pM, about 15 pM, about 20 pM, about 25 pM, or about 50 pM. Although certain concentrations are described above, they are exemplary and other concentrations may be used. Different nucleic acids may be present at different concentrations in the wells. Furthermore, all nucleic acids need not be present at the same concentration between wells.

[0035] FIG. 8 shows the ratio of read counts per mole in various wells of multiple assays.

[0036] Section 802 shows that the expected RT efficiency is 0.50, but various wells have RT efficiencies between 0.0 and 1.0. In particular, RT efficiencies below 0.39 indicate that the data is unreliable and can be excluded. RT efficiency indicates the effect of the reverse transcription portion of the RT-PCR process (e.g., what portion of the multiple nucleic acids (e.g., ssDNA spike-in) is converted by reverse transcription). A high RT efficiency indicates reliable data, and conversely, a low RT efficiency indicates that the data may need to be excluded. In some embodiments, the RT efficiency can be from about 0 to about 1. In some embodiments, the RT efficiency is about 0 to about 0.1, about 0 to about 0.2, about 0 to about 0.3, about 0 to about 0.4, about 0 to about 0.5, about 0 to about 0.6, about 0 to about 0.7, about 0 to about 0.8, about 0 to about 0.9, about 0 to about 0.95, about 0 to about 1, about 0.1 to about 0.2, about 0.1 to about 0.3, about 0.1 to about 0.4, about 0.1 to about 0.5, about 0.1 to about 0.6, about 0.1 to about 0 .7, about 0.1 to about 0.8, about 0.1 to about 0.9, about 0.1 to about 0.95, about 0.1 to about 1, about 0.2 to about 0.3, about 0.2 to about 0.4, about 0.2 to about 0.5, about 0.2 to about 0.6, about 0.2 to about 0.7, about 0.2 to about 0.8, about 0.2 to about 0.9, about 0.2 to about 0.95, about 0.2 to about 1, about 0.3 to about 0.4, about 0.3 to about 0.5, about 0.3 to about 0.6, about 0. 0.3 to about 0.7, about 0.3 to about 0.8, about 0.3 to about 0.9, about 0.3 to about 0.95, about 0.3 to about 1, about 0.4 to about 0.5, about 0.4 to about 0.6, about 0.4 to about 0.7, about 0.4 to about 0.8, about 0.4 to about 0.9, about 0.4 to about 0.95, about 0.4 to about 1, about 0.5 to about 0.6, about 0.5 to about 0.7, about 0.5 to about 0.8, about 0.5 to about 0.9, about 0.5 to about 0. 95, about 0.5 to about 1, about 0.6 to about 0.7, about 0.6 to about 0.8, about 0.6 to about 0.9, about 0.6 to about 0.95, about 0.6 to about 1, about 0.7 to about 0.8, about 0.7 to about 0.9, about 0.7 to about 0.95, about 0.7 to about 1, about 0.8 to about 0.9, about 0.8 to about 0.95, about 0.7 to about 1, about 0.8 to about 0.9, about 0.8 to about 0.95, about 0.8 to about 1, about 0.9 to about 0.95, about 0.9 to about 1, or about 0.95 to about 1.In some embodiments, the RT efficiency can be about 0, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 0.95, or about 1. In some embodiments, the RT efficiency can be at least about 0, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or about 0.95. In some embodiments, the RT efficiency can be up to about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 0.95, or about 1.

[0037] Section 804 shows that the expected DNase-RNA stability ratio is 1.00 to 2.00, but various wells have DNase-RNA stability ratios between 0.00 and 4.00. In particular, DNase stability ratios above 2.75 or below 1.75 indicate that the data is unreliable and can be excluded. The DNase-RNA stability ratio indicates how much of the nucleic acids added during the DNase step remain after transfer to another well (e.g., by comparing the amount of nucleic acid added during the step with the amount of nucleic acid remaining after the DNase step). A higher than expected DNase-RNA stability ratio indicates that data corruption may have occurred (e.g., data indicating that a later step was affected by the addition of nucleic acid), and conversely, a lower than expected DNase-RNA stability ratio indicates that the transfer was unsuccessful. In some embodiments, the DNase RNA stability ratio can be about 0 to about 2.5.In some embodiments, the DNase RNA stability ratio is about 0 to about 0.25, about 0 to about 0.5, about 0 to about 0.75, about 0 to about 1, about 0 to about 1.25, about 0 to about 1.5, about 0 to about 1.75, about 0 to about 2, about 0 to about 2.25, about 0 to about 2.5, about 0.25 to about 0.5, about 0.25 to about 0.75, about 0.25 to about 1, about 0.25 to about 1.25, about 0.2 5 to about 1.5, about 0.25 to about 1.75, about 0.25 to about 2, about 0.25 to about 2.25, about 0.25 to about 2.5, about 0.5 to about 0.75, about 0.5 to about 1, about 0.5 to about 1.25, about 0.5 to about 1.5, about 0.5 to about 1.75, about 0.5 to about 2, about 0.5 to about 2.25, about 0.5 to about 2.5, about 0.75 to about 1 , about 0.75 to about 1.25, about 0.75 to about 1.5, about 0.75 to about 1.75, about 0.75 to about 2, about 0.75 to about 2.25, about 0.75 to about 2.5, about 1 to about 1.25, about 1 to about 1.5, about 1 to about 1.75, about 1 to about 2, about 1 to about 2.25, about 1 to about 2.5, about 1.25 to about 1.5, about 1.25 to about 1.75 , about 1.25 to about 2, about 1.25 to about 2.25, about 1.25 to about 2.5, about 1.5 to about 1.75, about 1.5 to about 2, about 1.5 to about 2.25, about 1.5 to about 2.5, about 1.75 to about 2, about 1.75 to about 2.25, about 1.75 to about 2.5, about 2 to about 2.25, about 2 to about 2.5, or about 2.25 to about 2.5. In some embodiments, the DNase RNA stability ratio can be about 0, about 0.25, about 0.5, about 0.75, about 1, about 1.25, about 1.5, about 1.75, about 2, about 2.25, or about 2.5. In some embodiments, the DNase RNA stability ratio may be at least about 0, about 0.25, about 0.5, about 0.75, about 1, about 1.25, about 1.5, about 1.75, about 2, or about 2.25. In some embodiments, the DNase RNA stability ratio may be up to about 0.25, about 0.5, about 0.75, about 1, about 1.25, about 1.5, about 1.75, about 2, about 2.25, or about 2.5. In some embodiments, the DNase RNA stability ratio may be from about 1.75 to about 2.75.In some embodiments, the DNase RNA stability ratio can be about 1.75 to about 2, about 1.75 to about 2.25, about 1.75 to about 2.5, about 1.75 to about 2.75, about 2 to about 2.25, about 2 to about 2.5, about 2 to about 2.75, about 2.25 to about 2.5, about 2.25 to about 2.75, or about 2.5 to about 2.75. In some embodiments, the DNase RNA stability ratio can be about 1.75, about 2, about 2.25, about 2.5, or about 2.75. In some embodiments, the DNase RNA stability ratio can be at least about 1.75, about 2, about 2.25, or about 2.5. In some embodiments, the DNase RNA stability ratio can be up to about 2, about 2.25, about 2.5, or about 2.75. In some embodiments, the DNase RNA stability ratio can be about 2.75 to about 4. In some embodiments, the DNase RNA stability ratio can be about 2.75 to about 3, about 2.75 to about 3.25, about 2.75 to about 3.5, about 2.75 to about 3.75, about 2.75 to about 4, about 3 to about 3.25, about 3 to about 3.5, about 3 to about 3.75, about 3 to about 4, about 3.25 to about 3.5, about 3.25 to about 3.75, about 3.25 to about 4, about 3.5 to about 3.75, about 3.5 to about 4, or about 3.75 to about 4. In some embodiments, the DNase RNA stability ratio can be about 2.75, about 3, about 3.25, about 3.5, about 3.75, or about 4. In some embodiments, the DNase RNA stability ratio can be about 2.75, about 3, about 3.25, about 3.5, or about 3.75. In some embodiments, the DNase RNA stability ratio may be up to about 3, about 3.25, about 3.5, about 3.75, or about 4. In some embodiments, the DNase RNA stability ratio may be from about 2 to about 20.In some embodiments, the DNase RNA stability ratio is from about 2 to about 4, from about 2 to about 6, from about 2 to about 8, from about 2 to about 10, from about 2 to about 12, from about 2 to about 14, from about 2 to about 16, from about 2 to about 18, from about 2 to about 20, from about 4 to about 6, from about 4 to about 8, from about 4 to about 10, from about 4 to about 12, from about 4 to about 14, from about 4 to about 16, from about 4 to about 18, from about 4 to about 20, from about 6 to about 8, from about 6 to about 10, from about 6 to about 12, from about 6 to about 14, from about 6 to about 16, from about 6 to about 18, from about 6 to about 20, about 8 to about 10, about 8 to about 12, about 8 to about 14, about 8 to about 16, about 8 to about 18, about 8 to about 20, about 10 to about 12, about 10 to about 14, about 10 to about 16, about 10 to about 18, about 10 to about 20, about 12 to about 14, about 12 to about 16, about 12 to about 18, about 12 to about 20, about 14 to about 16, about 14 to about 18, about 14 to about 20, about 16 to about 18, about 16 to about 20, or about 18 to about 20. In some embodiments, the DNase RNA stability ratio can be about 2, about 4, about 6, about 8, about 10, about 12, about 14, about 16, about 18, or about 20. In some embodiments, the DNase RNA stability ratio may be at least about 2, about 4, about 6, about 8, about 10, about 12, about 14, about 16, or about 18. In some embodiments, the DNase RNA stability ratio may be up to about 4, about 6, about 8, about 10, about 12, about 14, about 16, about 18, or about 20.

[0038] Section 806 shows that the expected DNase inefficiency is about 0.05, but the DNase inefficiency is between 0.00 and 0.75 in various wells. In particular, a DNase inefficiency higher than 0.15 indicates that the data is unreliable and can be excluded. The DNase inefficiency indicates the effect of the DNase step by comparing the remaining amount of nucleic acid (e.g., DNA spike-in) added during the lysis step with the amount of nucleic acid (e.g., RNA spike-in) added during the DNase step. A higher DNase inefficiency indicates that the DNase step was not successful and the associated data should be excluded. In some embodiments, the DNase inefficiency can be between about 0 and about 0.1.In some embodiments, DNase inefficiency is from about 0 to about 0.01, from about 0 to about 0.02, from about 0 to about 0.03, from about 0 to about 0.04, from about 0 to about 0.05, from about 0 to about 0.06, from about 0 to about 0.07, from about 0 to about 0.08, from about 0 to about 0.09, from about 0 to about 0.1, from about 0.01 to about 0.02, from about 0.01 to about 0.03, from about 0.01 to about 0.04, from about 0.01 to about 0.05, from about 0. 01 to about 0.06, about 0.01 to about 0.07, about 0.01 to about 0.08, about 0.01 to about 0.09, about 0.01 to about 0.1, about 0.02 to about 0.03, about 0.02 to about 0.04, about 0.02 to about 0.05, about 0.02 to about 0.06, about 0.02 to about 0.07, about 0.02 to about 0.08, about 0.02 to about 0.09, about 0.02 to about 0.1, about 0.03 to about 0.04 , about 0.03 to about 0.05, about 0.03 to about 0.06, about 0.03 to about 0.07, about 0.03 to about 0.08, about 0.03 to about 0.09, about 0.03 to about 0.1, about 0.04 to about 0.05, about 0.04 to about 0.06, about 0.04 to about 0.07, about 0.04 to about 0.08, about 0.04 to about 0.09, about 0.04 to about 0.1, about 0.05 to about 0.06, about 0.05 to about It can be about 0.07, about 0.05 to about 0.08, about 0.05 to about 0.09, about 0.05 to about 0.1, about 0.06 to about 0.07, about 0.06 to about 0.08, about 0.06 to about 0.09, about 0.06 to about 0.1, about 0.07 to about 0.08, about 0.07 to about 0.09, about 0.07 to about 0.1, about 0.08 to about 0.09, about 0.08 to about 0.1, or about 0.09 to about 0.1. In some embodiments, the DNase inefficiency can be about 0, about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, or about 0.1. In some embodiments, DNase inefficiency can be at least about 0, about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, or about 0.09. In some embodiments, DNase inefficiency can be at most about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, or about 0.1.In some embodiments, DNase inefficiency can be from about 0 to about 1. In some embodiments, DNase inefficiency can be from about 0 to about 0.1, from about 0 to about 0.2, from about 0 to about 0.3, from about 0 to about 0.4, from about 0 to about 0.5, from about 0 to about 0.6, from about 0 to about 0.7, from about 0 to about 0.8, from about 0 to about 0.9, from about 0 to about 1, from about 0.1 to about 0.2, from about 0.1 to about 0.3, from about 0.1 to about 0.4, from about 0.1 ... to about 0.5, about 0.1 to about 0.6, about 0.1 to about 0.7, about 0.1 to about 0.8, about 0.1 to about 0.9, about 0.1 to about 1, about 0.2 to about 0.3, about 0.2 to about 0.4, about 0.2 to about 0.5, about 0.2 ...2 to about 0.7, about 0.2 to about 0.8, about 0.2 to about 0.9, about 0.2 to about 1, about 0.3 to about 0.4, about 0.3 to about 0.5, about 0.3 to about 0.6, about 0.3 to about 0.7, about 0.3 to about 0.8, about 0.3 to about 0.9, about 0.3 to about 1, about 0.4 to about 0.5, about 0.4 to about 0.6, about 0.4 to about 0.7, about 0.4 to about 0.8, about 0.4 to about 0.9, about 0.4 to about 1, about 0.5 to about 0.6, about 0. DNase inefficiency can be about 0.5 to about 0.7, about 0.5 to about 0.8, about 0.5 to about 0.9, about 0.5 to about 1, about 0.6 to about 0.7, about 0.6 to about 0.8, about 0.6 to about 0.9, about 0.6 to about 1, about 0.7 to about 0.8, about 0.7 to about 0.9, about 0.7 to about 1, about 0.8 to about 0.9, about 0.8 to about 1, or about 0.9 to about 1. In some embodiments, DNase inefficiency can be about 0, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or about 1. In some embodiments, DNase inefficiency may be at least about 0, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, or about 0.9. In some embodiments, DNase inefficiency may be at most about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or about 1.

[0039] Section (808) shows that the expected lytic RNA stability ratio is 0.20 to 0.50, but the lytic RNA stability ratio is 0.00 to 1.00 in various wells. In particular, a lytic RNA stability ratio below 0.15 indicates that the data is unreliable and can be excluded. The lytic RNA stability ratio indicates the effect of transfer between steps or whether the presence of RNase affects a later step by comparing the amount of nucleic acid (e.g., RNA spike-in) added during the lysis step with the amount of nucleic acid (e.g., other RNA spike-in) added during the DNase step. A higher than expected lytic RNA stability ratio indicates that data corruption may have occurred (e.g., data indicating that a later step was affected by the addition of nucleic acid), and conversely, a lower than expected lytic RNA stability ratio indicates that the transfer was unsuccessful. In some embodiments, the lytic RNA stability ratio can be about 0 to about 1.In some embodiments, the lytic RNA stability ratio is about 0 to about 0.1, about 0 to about 0.2, about 0 to about 0.3, about 0 to about 0.4, about 0 to about 0.5, about 0 to about 0.6, about 0 to about 0.7, about 0 to about 0.8, about 0 to about 0.9, about 0 to about 1, about 0.1 to about 0.2, about 0.1 to about 0.3, about 0.1 to about 0.4, about 0.1 from about 0.5, from about 0.1 to about 0.6, from about 0.1 to about 0.7, from about 0.1 to about 0.8, from about 0.1 to about 0.9, from about 0.1 to about 1, from about 0.2 to about 0.3, from about 0.2 to about 0.4, from about 0.2 to about 0.5, from about 0.2 to about 0.6, from about 0.2 to about 0.7, from about 0.2 to about 0.8, from about 0.2 to about 0.9, from about 0.2 to about 1, from about 0. 0.3 to about 0.4, about 0.3 to about 0.5, about 0.3 to about 0.6, about 0.3 to about 0.7, about 0.3 to about 0.8, about 0.3 to about 0.9, about 0.3 to about 1, about 0.4 to about 0.5, about 0.4 to about 0.6, about 0.4 to about 0.7, about 0.4 to about 0.8, about 0.4 to about 0.9, about 0.4 to about 1, about 0.5 to about 0.6, about 0. It can be about 0.5 to about 0.7, about 0.5 to about 0.8, about 0.5 to about 0.9, about 0.5 to about 1, about 0.6 to about 0.7, about 0.6 to about 0.8, about 0.6 to about 0.9, about 0.6 to about 1, about 0.7 to about 0.8, about 0.7 to about 0.9, about 0.7 to about 1, about 0.8 to about 0.9, about 0.8 to about 1, or about 0.9 to about 1. In some embodiments, the lytic RNA stability ratio can be about 0, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or about 1. In some embodiments, the lytic RNA stability ratio can be at least about 0, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, or about 0.9. In some embodiments, the lytic RNA stability ratio can be at most about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, or about 1.

[0040] Section (810) shows that the expected gDNA contamination ratio is 0.0 to 0.09, but the gDNA contamination ratio is 0.00 to 0.10 in various wells. In particular, a gDNA contamination ratio higher than 0.10 indicates that the data is unreliable and can be excluded. The gDNA contamination ratio indicates how much DNA was not converted over the course of the assay by comparing the multiple nucleic acids added during the lysis step (e.g., DNA spike-in) with the multiple nucleic acids added during the RT-PCR step prior to sequencing (e.g., another multiple DNA spike-in). A higher gDNA contamination ratio indicates that the addition of earlier nucleic acids may affect later steps and therefore the associated data may need to be excluded. In some embodiments, the gDNA contamination ratio may be about 0 to about 0.5.In some embodiments, the gDNA contamination ratio is from about 0 to about 0.05, from about 0 to about 0.1, from about 0 to about 0.15, from about 0 to about 0.2, from about 0 to about 0.25, from about 0 to about 0.3, from about 0 to about 0.35, from about 0 to about 0.4, from about 0 to about 0.45, from about 0 to about 0.5, from about 0.05 to about 0.1, from about 0.05 to about 0.15, from about 0.05 to about 0.2, from about 0.05 to about 0.25 , about 0.05 to about 0.3, about 0.05 to about 0.35, about 0.05 to about 0.4, about 0.05 to about 0.45, about 0.05 to about 0.5, about 0.1 to about 0.15, about 0.1 to about 0.2, about 0.1 to about 0.25, about 0.1 to about 0.3, about 0.1 to about 0.35, about 0.1 to about 0.4, about 0.1 to about 0.45, about 0.1 to about 0.5, about 0.15 to about 0.2 , about 0.15 to about 0.25, about 0.15 to about 0.3, about 0.15 to about 0.35, about 0.15 to about 0.4, about 0.15 to about 0.45, about 0.15 to about 0.5, about 0.2 to about 0.25, about 0.2 to about 0.3, about 0.2 to about 0.35, about 0.2 to about 0.4, about 0.2 to about 0.45, about 0.2 to about 0.5, about 0.25 to about 0.3, about 0.25 to about 0 0.35, about 0.25 to about 0.4, about 0.25 to about 0.45, about 0.25 to about 0.5, about 0.3 to about 0.35, about 0.3 to about 0.4, about 0.3 to about 0.45, about 0.3 to about 0.5, about 0.35 to about 0.4, about 0.35 to about 0.45, about 0.35 to about 0.5, about 0.4 to about 0.45, about 0.4 to about 0.5, or about 0.45 to about 0.5. In some embodiments, the gDNA contamination ratio can be about 0, about 0.05, about 0.1, about 0.15, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, or about 0.5. In some embodiments, the gDNA contamination ratio can be at least about 0, about 0.05, about 0.1, about 0.15, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, or about 0.45. In some embodiments, the gDNA contamination ratio can be at most about 0.05, about 0.1, about 0.15, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, or about 0.5.

[0041] While certain ratios are given and certain expected ratios are described, one of skill in the art would be able to determine the expected ratios based on expected read counts as well as determining the ratios from nucleic acid read counts. Additionally, certain parameters for unreliable data are described, but these are exemplary and may vary based on what steps are performed, under what conditions those steps are performed, and the ultimate experimental goals of the artisan.

[0042] Barcode Variable nucleotide sequences (barcodes) that function as indicators may be included in any of the reporter genes described herein.Furthermore, barcodes may be added in separate library preparation reactions.Variable nucleotide sequences described herein may be used as sample indexes to deconvolute the results obtained from the sequencing reactions used herein.

[0043] Once the contents of the cells have been released into their respective compartments by the lysis agent, the macromolecular components contained therein (e.g., macromolecular components of the sample such as RNA, DNA, or proteins) can be further processed within the compartment. In accordance with the methods and systems described herein, the macromolecular component contents of individual samples can be provided with unique identifiers so that upon characterization of those macromolecular components, they can be attributed as originating from the same sample or particle. The ability to attribute features to individual samples or groups of samples is provided by specifically assigning unique identifiers to individual samples or groups of samples. Unique identifiers, for example in the form of nucleic acid barcodes, can be assigned or associated to individual samples or populations of samples in order to tag or label the macromolecular components of the sample (and, consequently, their features) with unique identifiers. These unique identifiers can then be used to attribute sample components or features to individual samples or groups of samples.

[0044] In some aspects, this is done by dividing individual samples or groups of samples into compartments with unique identifiers or barcodes that contain unique molecular identifier sequences (UMIs). In some aspects, the unique identifiers are provided in the form of nucleic acid molecules (e.g., oligonucleotides) that contain nucleic acid barcode sequences that can bind to or otherwise associate with the nucleic acid content of the individual samples, or other components of the samples, particularly fragments of their nucleic acids. The nucleic acid molecules are divided into compartments such that between the nucleic acid molecules in a given compartment, the nucleic acid barcode sequences contained therein are the same, but between different compartments, the nucleic acid molecules can and do have different barcode sequences, or at least represent many different barcode sequences in all of the compartments in a given analysis. In some aspects, only one nucleic acid barcode sequence may be associated with a given compartment, but in some embodiments, there may be two or more different barcode sequences.

[0045] A nucleic acid barcode sequence may comprise from about 6 to about 20 or more nucleotides within the sequence of a nucleic acid molecule (e.g., an oligonucleotide). A nucleic acid barcode sequence may comprise from about 6 to about 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more nucleotides. In some embodiments, the length of the barcode sequence may be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides. In some embodiments, the length of the barcode sequence may be at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides. In some embodiments, the length of the barcode sequence may be up to about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or less nucleotides. These nucleotides may be completely contiguous, i.e., a single stretch of adjacent nucleotides, or may be separated into two or more separate subsequences separated by one or more nucleotides. In some embodiments, the separate barcode subsequences may be about 4 to about 16 nucleotides in length. In some embodiments, the barcode subsequences may be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some embodiments, the barcode subsequences may be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some embodiments, the barcode subsequences may be up to about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or shorter.

[0046] The nucleic acid molecules compartmentalized together can also contain other functional sequences useful for processing the nucleic acids from the samples compartmentalized together. These sequences include, for example, targeting or random / universal amplification primer sequences for amplifying genomic DNA from individual samples in the compartments while binding the associated barcode sequence, sequencing primers or primer recognition sites, hybridization or probe sequences for identifying the presence of a sequence or for pulling down the barcode nucleic acid, or any of many other potential functional sequences. Other mechanisms for compartmentalizing the oligonucleotides together may also be employed, including, for example, the combination of two or more compartments, one of which contains the oligonucleotide, or the microdispersion of the oligonucleotide into the compartments, for example, in a microfluidic system. In some embodiments, the primer comprises a barcode oligonucleotide. In some embodiments, the primer sequence is a targeting primer sequence that is complementary to a sequence in the template nucleic acid molecule. In some embodiments, the first nucleic acid molecule further comprises one or more functional sequences and the second nucleic acid molecule comprises one or more functional sequences. In some embodiments, the one or more functional sequences are selected from the group consisting of an adapter sequence, an additional primer sequence, a primer annealing sequence, a sequencing primer sequence, a sequence configured to bind to a flow cell of a sequencer, and a unique molecular identifier sequence.

[0047] For example, the above-mentioned barcoded nucleic acid molecules (e.g., barcoded oligonucleotides) are added to the sample. In some embodiments, the compartments include barcoded oligonucleotides having the same barcode sequence. In some embodiments, one compartment of the plurality of compartments includes barcoded oligonucleotides having the same barcode sequence, and each compartment in the plurality of compartments includes a unique barcode sequence. In some embodiments, the population of barcoded oligonucleotides provides a diverse barcode sequence library including at least about 1,000 different barcode sequences, at least about 5,000 different barcode sequences, at least about 10,000 different barcode sequences, at least about 50,000 different barcode sequences, at least about 100,000 different barcode sequences, at least about 1,000,000 different barcode sequences, at least about 5,000,000 different barcode sequences, or at least about 10,000,000 or more different barcode sequences. Furthermore, each barcoded oligonucleotide can be provided with a number of nucleic acid (e.g., oligonucleotide) molecules attached thereto. In particular, the number of molecules of a nucleic acid molecule comprising a barcode sequence on an individual barcoded oligonucleotide can be at least about 1,000 nucleic acid molecules, at least about 5,000 nucleic acid molecules, at least about 10,000 nucleic acid molecules, at least about 50,000 nucleic acid molecules, at least about 100,000 nucleic acid molecules, at least about 500,000 nucleic acid molecules, at least about 1,000,000 nucleic acid molecules, at least about 5,000,000 nucleic acid molecules, at least about 10,000,000 nucleic acid molecules, at least about 50,000,000 nucleic acid molecules, at least about 100,000,000 nucleic acid molecules, at least about 250,000,000 nucleic acid molecules, and in some embodiments, at least about 1 billion or more nucleic acid molecules. The nucleic acid molecules of a given barcoded oligonucleotide may comprise the same (or common) barcode sequence, different barcode sequences, or a combination of both. A given barcoded oligonucleotide nucleic acid molecule may comprise multiple sets of nucleic acid molecules. The nucleic acid molecules of a given set may comprise identical barcode sequences.The same barcode sequence can differ from the barcode sequence of another set of nucleic acid molecules.

[0048] Furthermore, when the population of barcoded oligonucleotides is partitioned, the resulting population of partitions may further comprise a diverse barcode library comprising at least about 1,000 different barcode sequences, at least about 5,000 different barcode sequences, at least about 10,000 different barcode sequences, at least about 50,000 different barcode sequences, at least about 100,000 different barcode sequences, at least about 1,000,000 different barcode sequences, at least about 5,000,000 different barcode sequences, or at least about 10,000,000 different barcode sequences. Further, each partition of the population may comprise at least about 1,000 nucleic acid molecules, at least about 5,000 nucleic acid molecules, at least about 10,000 nucleic acid molecules, at least about 50,000 nucleic acid molecules, at least about 100,000 nucleic acid molecules, at least about 500,000 nucleic acid molecules, at least about 1,000,000 nucleic acid molecules, at least about 5,000,000 nucleic acid molecules, at least about 10,000,000 nucleic acid molecules, at least about 50,000,000 nucleic acid molecules, at least about 100,000,000 nucleic acid molecules, at least about 250,000,000 nucleic acid molecules, and in some embodiments, at least about 1 billion nucleic acid molecules.

[0049] In some embodiments, it may be desirable to incorporate multiple different barcodes within a given compartment. For example, in some embodiments, the barcoded oligonucleotides within a compartment may include (1) a common barcode sequence shared by all barcoded oligonucleotides within the compartment, and (2) a unique molecular identifier or additional barcode sequence that differs between each barcoded oligonucleotide. The common barcode sequence may provide a stronger address or attribution of the barcode to a given compartment, for example, as a double or independent confirmation of the output from a given compartment, thereby making identification more certain in subsequent processing.

[0050] In some embodiments, barcoded oligonucleotides are attached to beads, where all of the nucleic acid molecules attached to a particular bead contain the same nucleic acid barcode sequence, but many different barcode sequences are represented across the population of beads used. In some embodiments, for example, hydrogel beads comprising a polyacrylamide polymer matrix are used as delivery vehicles for nucleic acid molecules to solid supports and compartments, as they can carry many nucleic acid molecules, and may be configured to release those nucleic acid molecules upon exposure to a particular stimulus, as described elsewhere herein.

[0051] The nucleic acid molecules (e.g., oligonucleotides) may be releasable from the beads upon application of a particular stimulus to the beads. In some embodiments, the stimulus may be a light stimulus, for example, by cleavage of a light-sensitive linkage releasing the nucleic acid molecule. In other embodiments, a thermal stimulus may be used, where an increase in the temperature of the bead environment causes cleavage of the linkage or other release of the nucleic acid molecule to form the bead. In still other embodiments, a chemical stimulus may be used that cleaves the linkage of the nucleic acid molecule to the bead or otherwise results in the release of the nucleic acid molecule from the bead. In one embodiment, such a composition comprises the polyacrylamide matrix described above for encapsulation of the sample, which may be degraded for release of the bound nucleic acid molecule via exposure to a reducing agent such as DTT.

[0052] The support that may be contemplated for use in the disclosed method may be, for example, a well, a matrix, a rod, a container, or a bead. The support may have any useful properties and characteristics, such as any useful size, surface chemistry, flowability, solidity, density, porosity, and composition. In some embodiments, the support is the surface of a well on a plate. In some embodiments, the support may be a bead, such as a gel bead. The bead may be solid or semi-solid. Further details of beads are provided elsewhere herein.

[0053] The support (e.g., bead) may include an anchor sequence (e.g., as described herein) functionalized thereon. The anchor sequence may be attached to the support, for example, via a disulfide bond. The anchor sequence may include a partial lead sequence and / or a flow cell functional sequence. Such sequences may allow sequencing of the nucleic acid molecules attached to the sequence by a sequencer (e.g., an Illumina sequencer). Different anchor sequences may be useful for different sequencing applications. The anchor sequence may include, for example, TruSeq or Nextera sequences. The anchor sequence may have any useful characteristic, such as any useful length and nucleotide composition. For example, the anchor sequence may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides. In some embodiments, the anchor sequence may include 15 nucleotides. The nucleotides of the anchor sequence may be naturally occurring or non-naturally occurring (e.g., as described herein). A bead may include multiple anchor sequences bound thereto. For example, a bead may include multiple first anchor sequences bound thereto. In some embodiments, a bead may include two or more different anchor sequences bound thereto. For example, a bead may include both multiple first anchor sequences (e.g., Nextera sequences) and multiple second anchor sequences (e.g., TruSeq sequences) bound thereto. For a bead that includes two or more different anchor sequences bound thereto, the sequence of each different anchor sequence may be distinguishable from the sequence of each of the other anchor sequences at the end distal to the bead. For example, the different anchor sequences may include one or more nucleotide differences in the 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides furthest from the bead.

[0054] In some embodiments, multiple different barcode molecules (e.g., nucleic acid barcode molecules) may be generated on the same support (e.g., beads). For example, two different barcode molecules may be generated on the same support. Alternatively, three or more different barcode molecules may be generated on the same support. Different barcode molecules attached to the same support may include one or more different sequences. For example, different barcode molecules may include one or more different barcode sequences and / or other sequences (e.g., starter sequences). In some embodiments, different barcode molecules attached to the same support may include the same barcode sequence. Different barcode molecules attached to the same support may include the same or different barcode sequences. Similarly, different barcode molecules may include the same or different unique molecular identifiers (UMIs).

[0055] Next-generation sequencing As described in the methods disclosed herein, sequencing of nucleic acid molecules is used and useful for detecting biological effects of test agents on cells, including cell-based assays. Generally, sequencing refers to methods and techniques for determining the sequence of nucleotide bases in one or more polynucleotides. Polynucleotides can be, for example, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including variants or derivatives thereof (e.g., single-stranded DNA). Sequencing can be performed by various systems currently available, such as, but not limited to, sequencing systems by Illumina, Pacific Biosciences, Oxford Nanopore, or Life Technologies (Ion Torrent). Such devices can provide a plurality of raw genetic data corresponding to the genetic information of a subject (e.g., a human) generated by the device from a sample provided by the subject. In some circumstances, the systems and methods provided herein may be used with proteomic information. Alternatively or additionally, sequencing may be performed using nucleic acid amplification, polymerase chain reaction (PCR) (e.g., digital PCR, quantitative PCR, or real-time PCR), or isothermal amplification. Such a system may provide a plurality of raw genetic data corresponding to the genetic information of a subject (e.g., a human) generated by the system from a sample provided by the subject. In some examples, such a system provides a sequencing read (also referred to herein as a "read"). A read may include a string of nucleic acid bases corresponding to the sequence of a sequenced nucleic acid molecule. In some circumstances, the systems and methods provided herein may be used with proteomic information.

[0056] Next generation sequencing includes many techniques (e.g., high-throughput sequencing) that can generate large amounts of sequence information, excluding Sanger sequencing or Maxam-Gilbert sequencing. Generally, next generation sequencing encompasses single molecule real-time sequencing, sequencing by synthesis, ion semiconductor sequencing, and the like. Exemplary next generation sequencing machines may include Ion Torrent machines from Thermo Fisher Scientific, such as the MiniSeq, iSeq100, NextSeq 1000, NextSeq 2000, NovaSeq 6000, NextSeq 550 series from Illumina, Inc., or the Sequel system from Pacific Biosciences.

[0057] Next generation sequencing machines used with the methods herein are capable of generating at least 1, 5, 10, 15, 25, 50, 75, 100, 200, or 300 or more gigabases of data from a single machine within 24 hours.

[0058] Next generation sequencing machines used with the methods herein are capable of generating data for at least 1, 1, 4, 10, 15, 25, 50, 75, 100, 200, 300, 500, or 1 billion or more sequence reads within 24 hours from a single machine.

[0059] Also included are computer programs, computing devices, or analysis platforms / systems for receiving and analyzing sequencing data, and then outputting one or more reports that can be sent or accessed electronically via a server, analysis portal, or email. The computing devices or analysis platforms can operate according to the algorithms and methods described herein.

[0060] Reaction mixture Also provided herein is a reaction mixture for determining the expression level of reporter in a sample by sequencing.In some embodiments, the reaction mixture comprises a control nucleic acid provided herein, at least a portion of the biological sample, and one or more enzymes or reagents sufficient to amplify barcodes in the sample, if present.

[0061] The control nucleic acid may be any one or more of single-stranded DNA, double-stranded DNA, single-stranded RNA, or double-stranded RNA. In some embodiments, the control nucleic acid is present at a concentration of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, or 500 copies per reaction mixture.

[0062] In some embodiments, the enzymes or reagents include reverse transcriptase, dNTPs, primer pairs specific for a barcode or control nucleic acid sequence, magnesium salts, or combinations thereof.

[0063] In some embodiments, the reaction mixture comprises one or more enzymes that can be used to amplify or replicate the control nucleic acid or the barcode nucleic acid. In some embodiments, the enzyme is a reverse transcriptase. Non-limiting examples of reverse transcriptase include, but are not limited to, avian myeloblastosis virus (AMV) reverse transcriptase, and Moloney murine leukemia virus (M-MuLV, MMLV), and mutants thereof.

[0064] In some embodiments, the reaction includes deoxynucleotide triphosphates (dNTPs). In some embodiments, the kit includes a mixture of each of the dNTPs required for amplification of the nucleic acid as well as any other desired nucleic acid (e.g., dATG, dCTP, dTTP, dGTP).

[0065] In some embodiments, the reaction mixture includes a magnesium salt. In some embodiments, the magnesium salt is included in an amount sufficient to allow an enzyme (e.g., reverse transcriptase) of the reaction to function and amplify the target nucleic acid. In some embodiments, the magnesium salt is magnesium chloride. In some embodiments, the reaction mixture includes a magnesium ion concentration of about 0.1 mM to about 50 mM. In some embodiments, the magnesium ion concentration is about 1 mM to about 10 mM.

[0066] In some embodiments, the volume of the reaction mixture is about 10 microliters to about 100 microliters. In some embodiments, the volume of the reaction mixture is about 20 microliters to about 90 microliters, about 30 microliters to about 80 microliters, or about 40 microliters to about 60 microliters. In some embodiments, the volume of the reaction mixture is about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 microliters.

[0067] Quality control method As mentioned above, non-limiting examples of assay designs according to the methods described herein may include the steps of: 1) seeding cells into wells of a well plate; 2) contacting the cells with a test agent and allowing time for the test agent to affect reporter activation (e.g., expression of a barcode) via the target polypeptide; 3) lysing the cells to release nucleic acids from the cells; 4) treating the lysate with DNAse to generate relevant RNA; 5) reverse transcribing and / or amplifying the reverse transcribed mRNA to generate cDNA or amplified cDNA (e.g., RT-PCR); 6) preparing the cDNA for a next generation sequencing reaction; and 7) sequencing the prepared cDNA.

[0068] FIG. 3 shows the read counts of multiple control nucleic acids added during the DNase step. In this embodiment shown, the multiple nucleic acids are multiple double-stranded DNA spikes. In this example, multiple nucleic acids were added during the lysis step. For assays where a DNase step was not performed, the read counts of the multiple nucleic acids in the left half of the graph labeled "spiked-no DNase" were obtained, and for assays where a DNase step was performed, the read counts in the right half of the graph labeled "spiked-standard-p" were obtained. Thus, the read counts in the left half of the graph are large (between 1e+05 and 1e+04) because multiple nucleic acids (e.g., double-stranded DNA spikes) were not cleaved and therefore still present in solution and could be identified. In contrast, the read counts on the left side are much lower because many of the multiple nucleic acids were cleaved and therefore still not present in solution and could not be identified. Therefore, the use of barcodes allows one to assess the extent of the DNase step (e.g., reducing the amount of double-stranded DNA from approximately 1e+05 to 0 reads), which can also be assessed using a certain ratio based on read counts (e.g., DNase inefficiency).

[0069] FIG. 4 shows the read percentage of the control nucleic acids relative to the original total amount of the nucleic acids introduced. In this embodiment shown, the nucleic acids are single-stranded DNA. Furthermore, in this embodiment shown, the nucleic acids were added just before the RT-PCR step. In this embodiment, the read percentages on the left side of the graph labeled "spike-no RT" are the read percentages of assays that did not use reverse transcription reagents. Thus, the read percentages tend to be high, many near 1.0, because in the RT-PCR step, none of the RNA in the sample is converted to DNA, and therefore the only reads that made it to the sequencing reaction are those associated with the ssDNA spike-in. The read percentages on the right side of the graph labeled "spike-standard-p" are the read percentages of assays that did undergo an RT-PCR step, and the read percentages were determined after the RT-PCR step. Thus, the read percentages indicate that most of the RNA present in the sample was transcribed into cDNA.

[0070] 5 shows the read counts of various nucleic acids introduced in 10,000 spike amounts added during one or more of the steps described above. For example, the read count labeled "n10185" represents the amount of RNA spike added during the RT-PCR step, the read count labeled "n10186" represents the amount of RNA added during the DNase step, the read count labeled "n10187" represents the amount of double-stranded DNA spike added during the lysis step, and the read count labeled "n10188" represents the amount of single-stranded DNA during the RT-PCR step. In this embodiment shown, the read count n10185 is taken at the beginning of the RT-PCR step, and therefore the read count is at or slightly below 10,000 because the reverse transcriptase has not yet been performed. In this embodiment shown, read count n10186 provides information about the preservation of RNA during the DNase step, and thus the read count is at or slightly below 10,000, indicating that DNase did not degrade the RNA spike. In this embodiment shown, read count n10187 provides information about the efficiency of DNA degradation in the DNase step, and thus the read count is lower because a lot of double-stranded DNA is cut during the DNase step. Furthermore, in this embodiment shown, read count n10188 provides information about the efficiency of the RT-PCR step, and thus the read count is lower because a lot of the sample RNA was converted to DNA, lowering the overall count of the added DNA spike.

[0071] 7 shows various read counts for RNA spikes during the DNase step (e.g., read count 702), DNA spikes during the lysis step (e.g., read count 704), RNA spikes during the lysis step (e.g., read count 706), DNA spikes during the RT-PCR step (e.g., read count 708), and RNA spikes during the RT-PCR step (e.g., read count 710). The read counts shown are associated with two different assays (e.g., run 110 and run 112) and are from multiple wells used in each assay (e.g., plate 105, plate 106, plate 109, plate 110, plate 5, and plate 6).

[0072] As mentioned above, from the above results shown in Figures 3-7, certain ratios may be calculated, such as lytic RNA stability ratio, DNase inefficiency, RT efficiency, RT-PCR RNA stability ratio, and DNase RNA stability ratio. EXAMPLES

[0073] The following illustrative examples represent embodiments of the compositions and methods described herein and are not meant to be limiting in any way.

[0074] Example 1 - RNA-based spiking in controls allows for the rejection of data from wells exhibiting fluid handling defects Special process controls were created that could inform the performance of our workflow at several critical steps. These controls consist of various synthetic DNA and RNA molecules that were added in precise amounts to the samples at various intermediate reactions. These "spike-ins" then undergo the same downstream process along with the actual samples. Sequencing readouts of these controls are performed at the end of the multiplexed assay, and the results provide important insight into how the process performed and, if there was a problem, which aspect of the process was experiencing the problem. These control metrics allowed for the rapid detection, investigation, and resolution of process issues.

[0075] For example, in Figure 1, an HTS assay was performed in which cells in wells of a multi-well plate were exposed to a test agent to express (or not express) a barcoded reporter. RNA was harvested, reverse transcribed, and sequenced using a next-generation sequencing assay. The performance of several synthetic RNA spikes provides insight into how to perform multiplexed assays. In the example in Figure 1, there are four plates that contain significant numbers of wells with low abundance of these molecules, indicating a process challenge in one of our liquid transfer steps. In this figure, wells with RNA spike-in read counts were below a predefined cutoff and were rejected.

[0076] In addition to measuring the impact of improvements realized by using separate quality control spike-ins, we developed analytical tools to measure the quality of the data. Most high-throughput bioassays are performed in multi-well microtiter plates. Typically, each well of these plates tests a specific experimental condition, i.e., the effect of a chemical on the cells in the well. In addition to the process controls mentioned above, the behavior of our cell reporter allows us to apply several specific quality control filters to distinguish poor from good performing wells. Only data coming from wells with good quality are passed to the downstream bioinformatics pipeline for analysis. Metrics on how many wells fail the QC gates and why allow us to monitor the process and troubleshoot problems more focused.

[0077] Example 2 - RNA-based spikes in controls allow for the rejection of data from wells exhibiting fluid handling defects Specific process controls are determined for the process including the lysis, DNase, RT-PCR, and sequencing steps as described above.

[0078] Lyse the cells by creating a solution containing 20 μL of lysis buffer and the plurality of cells, and add the DNA spike (e.g., the second plurality of nucleic acids) and the RNA spike (e.g., the first plurality of nucleic acids) such that the RNA spike (e.g., the first plurality of control nucleic acids) has a concentration of 0.5 pM and the DNA spike (e.g., the second plurality of control nucleic acids) has a concentration of 0.5 pM.

[0079] Transfer 8 µL of lysis solution to a new well and add 4 µM buffer (so that the total volume is 12 µL), as well as add an RNA spike-in (e.g., a fifth multiple nucleic acid) to achieve a concentration of 0.5 pM, and perform a DNase step.

[0080] Transfer 4 μL of DNase solution to a new well to add RNA spikes (e.g., third plurality of nucleic acids) to a total concentration of 0.13 pM and DNA spikes (e.g., fourth plurality of nucleic acids) to a total concentration of 0.13 pM (resulting in a total volume of 12 μL). RT-PCR is performed. Samples for the RT PCR step are then prepared and sequenced on a next generation sequencing machine. Total counts are collected for each plurality of nucleic acids after sequencing, and the data is used to determine if the assay is performing within a certain tolerance and accept or reject data from individual wells accordingly. Alternatively, if too many wells fail to pass, the operator may troubleshoot to determine which step is running inefficiently.

[0081] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous modifications, changes, and substitutions will occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be utilized in practicing the invention.

[0082] All publications, patent applications, issued patents, and other documents mentioned herein are incorporated by reference herein as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions contained in the texts incorporated by reference are excluded to the extent they conflict with definitions in this disclosure.

Claims

1. 1. A method for quality assurance for a high throughput screening system, comprising: (a) providing a first composition comprising a plurality of cells; (b) lysing the plurality of cells to obtain a lysed cell composition, the lysed cell composition comprising a first plurality of control nucleic acids, a second plurality of control nucleic acids, or a first plurality of control nucleic acids and a second plurality of control nucleic acids; (c) preparing a reverse transcription reaction comprising at least a portion of the lysed cell composition and a third plurality of control nucleic acids, a fourth plurality of control nucleic acids, or a third plurality of control nucleic acids and a fourth plurality of control nucleic acids; (d) reverse transcribing the reverse transcription reaction to obtain a reverse transcribed nucleic acid, and optionally amplifying the reverse transcribed nucleic acid; (e) preparing a sequencing library comprising the reverse transcribed nucleic acids; (f) performing a sequencing reaction on the sequencing library to obtain sequence information for (i) a plurality of reporters, and (ii) one or more of the first plurality of control nucleic acids, the second plurality of control nucleic acids, the third plurality of control nucleic acids, or the fourth plurality of control nucleic acids; A method comprising:

2. The method of claim 1, further comprising contacting the lysed cell composition with a DNA degrading agent.

3. 3. The method of claim 2, further comprising contacting the lysed cell composition with a fifth plurality of control nucleic acids prior to contacting the lysed cell composition with the DNA degrading agent.

4. The method of claim 1 , further comprising contacting the plurality of cells with a test agent.

5. The method of claim 4 , wherein the step of contacting the plurality of cells with the test agent occurs prior to step (b).

6. The method of any one of claims 1 to 5, wherein the sequence information for the plurality of reporters provides information on the level of mRNA expression for at least one reporter of the plurality of reporters.

7. The method of claim 6 , wherein the at least one reporter comprises a barcode sequence.

8. 8. The method of claim 7, wherein the barcode sequence is operably linked to a promoter element and / or an enhancer element.

9. 9. The method of any one of claims 1 to 8, comprising performing a sequencing reaction on the sequencing library to obtain sequence information for (i) a plurality of reporters, and (ii) the first plurality of control nucleic acids, the second plurality of control nucleic acids, and the fourth plurality of control nucleic acids.

10. The method of any one of claims 1 to 9, wherein the first plurality of control nucleic acids comprises RNA nucleic acids.

11. 11. The method of claim 10, wherein the first plurality of control nucleic acids is added to the plurality of cells prior to lysing the plurality of cells.

12. 12. The method of claim 10 or 11, wherein the first plurality of control nucleic acids comprises a first nucleotide sequence, a second nucleotide sequence, a third nucleotide sequence, and / or a fourth nucleotide sequence that are distinguishable from one another.

13. 13. The method of any one of claims 1 to 12, further comprising normalizing sequence information for at least one reporter of the plurality of reporters based on the first plurality of control nucleic acids.

14. The method of any one of claims 1 to 13, wherein the second plurality of control nucleic acids comprises double-stranded DNA nucleic acids.

15. The method of any one of claims 1 to 14, wherein the third plurality of control nucleic acids comprises RNA nucleic acids.

16. 16. The method of any one of claims 1 to 15, further comprising determining the relative abundance of the second plurality of control nucleic acids within the reverse transcribed nucleic acids.

17. The method of any one of claims 1 to 16, wherein the fourth plurality of control nucleic acids comprises single-stranded DNA nucleic acids.

18. 18. The method of any one of claims 1 to 17, further comprising determining a control sequence that is not a sequence of the plurality of reporters, the first plurality of control nucleic acids, the second plurality of control nucleic acids, and the fourth plurality of control nucleic acids.

19. 19. The method of any one of claims 1 to 18, wherein the plurality of reporters comprises at least 10 reporters, at least 100 reporters, at least 1,000 reporters, at least 5,000 reporters, at least 10,000 reporters.

20. 20. The method of claim 19, wherein the at least 10 reporters, the at least 100 reporters, the at least 1,000 reporters, the at least 5,000 reporters, or the at least 10,000 reporters each comprise a unique barcode sequence.

21. 21. The method of claim 19 or 20, wherein each of the at least 10 reporters, the at least 100 reporters, the at least 1,000 reporters, the at least 5,000 reporters, or the at least 10,000 reporters uniquely identifies a target molecule.

22. 22. The method of claim 21, wherein the target molecule is a polypeptide expressed from a nucleic acid by the plurality of cells.

23. 23. The method of any one of claims 1 to 22, comprising altering the nucleotide sequence of any of the first plurality of control nucleic acids, the second plurality of control nucleic acids, the third plurality of control nucleic acids, the fourth plurality of control nucleic acids, or the fifth plurality of control nucleic acids for a subsequent experimental run.

24. The method of any one of claims 1 to 23, wherein the sequencing reaction is a high-throughput sequencing reaction.

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