Linked dual barcode insert construct

Abstract

Contemporary gene sequencing technologies, including "next generation sequencing" technologies, can include sequencing multiple fragments of a target polynucleotide. However, the limitations of existing sequencing technologies, as well as the often repeated or otherwise difficult-to-sequence structures of natural polynucleotides, mean that generating accurate sequences may be difficult and / or expensive. The methods provided herein include inserting a double polynucleotide "barcode" into a target polynucleotide together with an adjacent primer sequence before other sequencing processes. These inserted barcodes can improve the accuracy of the sequence generated for the target by adding "noise" to the target, thereby allowing subsequent sequencing technologies (e.g., alignment, stitching, etc.) to more accurately estimate the "target plus barcode" sequence. Primers can cause the fragment to start at a point corresponding to the start of other sequences within the target, making it easy to stitch the sequence ends together. The barcode can then be removed to provide the sequence of the target polynucleotide.

Description

Technical Field

[0001] The present disclosure relates to methods and apparatus for polynucleotide barcode insertion and sequencing. Background Art

[0002] Early DNA sequencing techniques, such as the chain termination method, provided a reliable solution for reading single DNA fragments. See F. Sanger et al. (1977), DNA sequencing with chain-terminating inhibitors, Proc. Nat. Acad. Sci. 74, 5463-5467. Although these first generation technologies are effective for sequencing target genes, applying them to the sequencing of entire chromosomes or genomes is costly and expensive. For example, the first sequencing of the human genome—completed using the Sanger method—cost hundreds of millions of dollars and took more than 10 years to complete. This high cost is primarily due to the sequential nature of the first generation sequencing methods; each fragment must be read individually and manually assembled to construct a complete genome.

[0003] Next Generation Sequencing (NGS) technology has significantly reduced the cost of DNA sequencing by parallelizing the reading of DNA fragments. Some NGS methods are able to perform millions of sequence reads simultaneously, generating millions of base pairs of data in a few hours. See N. Hall (2007), Advanced sequencing technologies and their wider impact in microbiology, The Journal of Experimental Biology, 209, 1518-1525. Many NGS technologies have been proposed, which use various chemical processes, use different read lengths, and have demonstrated various ranges of accuracy. See M. Metzker (2010), Sequencing technologies—the next generation, Nature Reviews, Genetics, Vol. 11, 31-46; see also J. Shen Dulei et al. (2008), Next-generation DNA sequencing, Nature Reviews, Biotechnology, Vol. 26, No. 10, 1135-1145.

[0004] NGS methods typically involve separating a DNA sample into fragments and reading the nucleotide sequences of these fragments in parallel. The resulting data generated from this process includes read data for each of these fragments, which contains a continuous sequence of nucleotide base pairs (G, A, T, C). However, although the arrangement of base pairs in a given fragment read is known, the arrangement of the fragment reads relative to each other is not known. Therefore, to determine the sequence of a larger DNA chain (such as a gene or chromosome), the read data from multiple fragments must be aligned. This alignment is relative to other read fragments and may include overlapping fragments, depending on the specific NGS method used. Some NGS methods use computational techniques and software tools to perform read data alignment.

[0005] Accurate sequence read alignment is the first step in identifying genetic variants in a sample genome. The diversity of genetic variants can cause alignment algorithms and techniques to align sequence reads to incorrect locations in the genome. In addition, the reading process used to generate sequence reads can be complex and prone to errors. As a result, many sequence read alignment technologies can make sequence reads inconsistent within a genome, which can lead to incorrect detection of variants in subsequent analyses.

[0006] Once the read data is aligned, the aligned data can be analyzed to determine the nucleotide sequence of a gene locus, a gene, or an entire chromosome. However, the nucleotide value differences between the overlapping read fragments may indicate variants (such as single nucleotide polymorphisms (SNPs) or insertions or deletions (INDELs) and other possible variants). For example, if the read fragments overlapping at a specific locus are different, these differences may indicate the presence of heterozygous SNPs. As another example, if the overlapping read fragments are identical at a single nucleotide, but different from a reference genome, the gene locus or gene may be a homozygous SNP relative to the reference genome. Accurately determining such variants is an important aspect of genome sequencing because these variants may represent mutations, genes that cause specific diseases, and / or be used to determine genotypes for specific DNA samples.

[0007] In recent years, the demand for efficient and low-cost DNA sequencing has continued to increase. Although NGS technology has significantly improved upon first-generation technologies, the high degree of parallelism of NGS technology presents challenges not encountered in earlier sequencing technologies. Errors in the reading process can adversely affect the alignment of the resulting read data and may subsequently lead to inaccurate sequence determination. In addition, reading errors may lead to the false detection of variants.

[0008] A more complete and accurate understanding of the entire human genome and individual genomes can improve medical diagnostics and treatments. NGS technology has reduced the time and cost of sequencing an individual's genome, which provides the potential for major improvements in medicine and genetics in areas that were not previously feasible. Understanding genetic variation among humans provides a framework for understanding genetic disorders and Mendelian diseases. However, discovering these genetic variants depends on reliable read data and accurate read sequence alignment. Summary of the invention

[0009] In a first aspect, a method is provided, comprising: (i) adding a probe to a sample comprising a target polynucleotide, wherein the probe comprises a payload polynucleotide and an insertion vector, wherein the payload polynucleotide comprises a first polynucleotide barcode, a reverse primer, a forward primer, and a second polynucleotide barcode, and wherein the insertion vector inserts the payload polynucleotide into the target polynucleotide; and (ii) applying an amplifier to generate: (1) a first copy of the target polynucleotide, wherein the payload polynucleotide is inserted, starting from the reverse primer, such that the first copy comprises a complement of the first polynucleotide barcode near the end of the first copy, and (2) a second copy of the target polynucleotide, wherein the payload polynucleotide is inserted, starting from the forward primer, such that the second copy comprises a complement of the second polynucleotide barcode near the end of the second copy.

[0010] The insertion vector may include a Tn5 transposase.The insertion vector may include CRISPR-Cas9.

[0011] After the probe is added to the sample, at least a portion of the sample can be sequenced multiple times to obtain a read length of a first copy of the target polynucleotide and a read length of a second copy of the target polynucleotide. The sequence of the target polynucleotide can be determined based on the read length of the first copy and the read length of the second copy. Determining the sequence of the target polynucleotide may include determining a preliminary sequence of the target polynucleotide based on the read length of the first copy and the read length of the second copy, and removing the sequence of the first polynucleotide barcode and the second polynucleotide barcode from the preliminary sequence. Determining the preliminary sequence may include stitching the ends of the first copy together with the ends of the second copy.

[0012] The probe can be sequenced to generate a probe sequence, and based on the probe sequence, the corresponding sequences of the first polynucleotide barcode and the second polynucleotide barcode can be determined. It can be determined that the first polynucleotide barcode and the second polynucleotide barcode correspond to each other.

[0013] Stitching the ends of the first copy together with the ends of the second copy can be performed in response to determining that the first copy includes a determined sequence of a first polynucleotide barcode near an end of the first copy and the second copy includes a determined sequence of a second polynucleotide barcode near an end of the second copy.

[0014] After administering the amplifier, an additional plurality of polynucleotide barcodes can be inserted into the target polynucleotide to obtain additional copies of the target polynucleotide in which the additional plurality of polynucleotide barcodes are inserted. After inserting the additional plurality of polynucleotide barcodes into the target polynucleotide, at least a portion of the sample can be sequenced multiple times to obtain reads of the additional copies of the target polynucleotide. Determining the sequence of the target polynucleotide can include determining the sequence based on the reads of the additional copies of the target polynucleotide.

[0015] The method may include: before adding the probe to the sample, sequencing at least a portion of the sample multiple times to obtain multiple unmodified reads of the target polynucleotide, wherein removing the first polynucleotide barcode and the second polynucleotide barcode from the preliminary sequence includes comparing the preliminary sequence to at least one of the multiple unmodified reads of the target polynucleotide.

[0016] The target polynucleotide may comprise DNA. The target polynucleotide may comprise RNA. The target polynucleotide may be a first isoform of an RNA sequence, and the sample may comprise a second isoform of the RNA sequence. The first isoform may be different from the second isoform.

[0017] The first polynucleotide barcode and the second polynucleotide barcode can have the same sequence.The second polynucleotide barcode can be a reverse complementary version of the first polynucleotide barcode.

[0018] In another aspect, a non-transitory computer-readable medium having stored therein instructions executable by a computing device to cause the computing device to determine the sequence of a target polynucleotide according to the method described herein is provided.

[0019] In yet another aspect, a probe is provided, comprising: (i) a payload polynucleotide, wherein the payload polynucleotide comprises a first polynucleotide barcode, a reverse primer, a forward primer, and a second polynucleotide barcode; and (ii) an insertion vector, wherein the insertion vector is configured to insert the payload polynucleotide into a target polynucleotide.

[0020] In yet another aspect, a method for generating a payload polynucleotide is provided. The method comprises: (i) obtaining a template polynucleotide comprising a first polynucleotide barcode and a first amplification primer; (ii) forming a complementary polynucleotide on the template polynucleotide, such that the complementary polynucleotide comprises a second amplification primer and a second polynucleotide barcode; (iii) forming a hairpin between the template polynucleotide and the complementary polynucleotide; and (iv) dissociating the complementary polynucleotide from the template polynucleotide via a denaturation process.

[0021] The insertion vector may comprise a Tn5 transposase.The insertion vector may comprise a CRISPR-Cas9.

[0022] The foregoing summary is illustrative only and is not limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1A Exemplary polynucleotides are shown.

[0024] Figure 1B Exemplary polynucleotides are shown.

[0025] Figure 1C Exemplary polynucleotides are shown.

[0026] Figure 2A Aspects of exemplary methods for improving polynucleotide sequencing by inserting additional known sequences into a target polynucleotide are shown.

[0027] Figure 2B Aspects of exemplary methods for improving polynucleotide sequencing by inserting additional known sequences into a target polynucleotide are shown.

[0028] Figure 2C Aspects of exemplary methods for improving polynucleotide sequencing by inserting additional known sequences into a target polynucleotide are shown.

[0029] Figure 2D Aspects of exemplary methods for improving polynucleotide sequencing by inserting additional known sequences into a target polynucleotide are shown.

[0030] Figure 2E Aspects of exemplary methods for improving polynucleotide sequencing by inserting additional known sequences into a target polynucleotide are shown.

[0031] Figure 3 An exemplary alignment of two nucleotide sequences is shown.

[0032] Figure 4A An exemplary identification of a known sequence that has been inserted into two target polynucleotides is shown.

[0033] Figure 4B An exemplary identification of a known sequence that has been inserted into a target polynucleotide is shown.

[0034] Figure 5A Examples of polynucleotides that can be inserted into a target polynucleotide are illustrated.

[0035] Figure 5B Examples of polynucleotides that can be inserted into a target polynucleotide are shown.

[0036] Figure 6 Examples of polynucleotides that have been inserted into a target polynucleotide and exemplary read sequences generated therefrom are shown.

[0037] Figure 7 The alignment of the two sequences is shown.

[0038] Fig. 8A Aspects of an exemplary process for developing a double-linked insert sequence library are shown.

[0039] Figure 8B Aspects of an exemplary process for developing a double-linked insert sequence library are shown.

[0040] Figure 8C Aspects of an exemplary process for developing a double-linked insert sequence library are shown.

[0041] Fig.8D Aspects of an exemplary process for developing a double-linked insert sequence library are shown.

[0042] Fig. 9 A flow chart of an exemplary method is shown.

[0043] Fig.10 A flow chart of an exemplary method is shown.

[0044] Fig.11 A flow chart of an exemplary method is shown. DETAILED DESCRIPTION

[0045] The following detailed description describes various features and functions of the disclosed systems and methods with reference to the accompanying drawings. The illustrative system and method embodiments described herein are not meant to be limiting. It can be easily understood that certain aspects of the disclosed systems and methods can be arranged and combined in a variety of different configurations, all of which are considered herein.

[0046] I. Introduction

[0047] Next-generation sequencing (NGS) has significantly reduced the time and cost required to sequence an entire genome. Previous techniques involved reading out DNA fragments sequentially and having trained biochemists align the reads to determine the sequence of an entire chromosome. NGS technology parallelizes the sequencing process, allowing millions of DNA fragments to be read simultaneously. Automated computational analysis then attempts to align the reads to determine the nucleotide sequence of a locus, gene, chromosome, or entire genome.

[0048] The increasing popularity of NGS technology has generated a large amount of genomic data. Analysis of genomic data from single and multiple samples can provide meaningful insights into the genetics of a sample (e.g., a single human patient) or a species. Variations between genomes may correspond to different traits or diseases within a species. Variations may take the form of single nucleotide polymorphisms (SNPs), insertions and deletions (INDELs), and structural differences in the DNA itself, such as copy number variants (CNVs) and chromosome rearrangements. By studying these variations, scientists and researchers can better understand differences within species, the causes of certain diseases, and provide patients with better clinical diagnoses and personalized medicines.

[0049] The quality and accuracy of genomic datasets are critical for subsequent analyses and studies performed on these datasets. However, flaws in the NGS technologies used to generate these genomic datasets can lead to errors in the reading process itself and in the alignment of the read data, resulting in uncertainty in the (multiple) output sequences. If the NGS machine incorrectly reads the nucleobase and records it in the read data, subsequent analysis may incorrectly identify variants at that locus. Incorrect variant detection may also occur if the alignment of the read data is inaccurate. In addition or alternatively, an incorrect number of repeat sequences may be detected, or indels may be incorrectly detected or ignored. If these sources of error are not accounted for, false-positive variant detection may lead to incorrect clinical diagnosis or the discovery of non-existent variants.

[0050] To mitigate these errors, some NGS analysis pipelines include filtering steps to detect and discard false positive variant calls. As used herein, "variant calling" can be used to refer to variant detection. Some filtering techniques employ hard filters that analyze one or more aspects of a variant call, compare it to one or more criteria, and provide a determination as to whether it is a true positive variant call or a false positive variant call. For example, if multiple read fragments aligned at a particular locus show three or more different bases, a hard filter may determine that a variant call is a false positive.

[0051] Other filtering techniques use statistical or probabilistic models and may involve performing statistical inference based on one or more manually selected variables of variant calls. Variant calls may include a set of read data of DNA fragments aligned with each other. Each DNA fragment read data may include metadata specifying the confidence level of the read accuracy (i.e., the quality of the bases), information about the process used to read the DNA fragments, and other information. DNA sequencing experts may select features that they think may distinguish true positive and false positive variant calls. Then, a set of labeled examples (e.g., known true variant calls and quantitative values ​​of manually selected features) can be used to train a statistical model (e.g., a Bayesian mixed model). Once trained, new variant calls can be provided to the statistical model, which can determine the confidence level indicating how likely it is that the variant call is a false positive.

[0052] False positive variant calls can be avoided or mitigated by performing more accurate read sequence alignments, and / or by improving the robustness of the variant callers themselves. Some variant callers can detect SNPs and INDELs via local de-novo assembly of haplotypes. When such a variant caller encounters a region of read pileups that indicates a variant, the variant caller can attempt to reassemble or realign the sequence reads. By analyzing these realignments, these types of variant callers can assess the likelihood that the read pileup region contains a variant.

[0053] Many different read processes can be used to generate DNA fragment read data for a sample. Each read process may vary in read length, amplification method, materials used, and technology used (e.g., chain termination, ligation, etc.). The nature and source of errors may be different for each read process. Therefore, the features that distinguish incorrect alignment, invalid variant calls, or false positive variant detections may be different in different read processes.

[0054] Note that a "sample" can be a sample from a biological organism (e.g., a human, an animal, a plant, etc.) and / or can be a sample containing synthetic content. For example, a sample may contain synthetic DNA (or RNA, or some other synthetic polynucleotide) that is created, for example, to store information in a sequence or other property of the synthetic DNA. Thus, the methods described herein can be applied to extract information stored in such a sample.

[0055] II. Terminology

[0056] A. Next Generation Sequencing (NGS)

[0057] NGS generally refers to DNA sequencing technologies that involve sequencing multiple DNA fragments of a sample in parallel. The output data can include the nucleotide sequence of each read, which can then be assembled to form longer sequences within a gene, a whole gene, a chromosome, or a whole genome. The specific aspects of a particular NGS technology may vary depending on the sequencing instrument, vendor, and various other factors. Secondary analysis may then involve aligning / assembling the reads to generate predicted target sequences, detecting variants in the sample, etc.

[0058] An exemplary polynucleotide (e.g., DNA) sequencing pipeline can include polynucleotide sequencing (e.g., using one or more next-generation DNA sequencers), read data alignment, and variant calling. As described herein, a "pipeline" can refer to a combination of hardware and / or software that receives input material or data and generates a model or output data. An exemplary pipeline receives a sample containing polynucleotides as input, which is sequenced by (multiple) polynucleotide sequencers to output read data. Read data alignment is achieved by receiving raw input read data and generating aligned read data. Variant calling can then continue by analyzing the aligned read data and outputting potential variants.

[0059] The input sample can be a biological sample (e.g., biopsy material) taken from a specific organism (e.g., a human). The sample can be isolated DNA, RNA, or some other polynucleotides, and can contain a single gene, a gene cluster, a whole chromosome, or an entire genome. The polynucleotides of interest in the sample can include natural or artificial DNA, RNA, or other polynucleotides formed by some other types of nucleotides and / or combinations of these types of nucleotides. The sample may include substances or DNA isolated from two or more cells in a specific organism. When the sample contains RNA, it may contain multiple different isoforms of a specific RNA sequence (e.g., various different isoforms involving folded RNA, proteins generated from RNA by ribosomes or (multiple) other structures, or some other RNA-related substances).

[0060] (Multiple) polynucleotide sequencers may include any scientific instrument that autonomously or semi-autonomously performs polynucleotide sequencing (e.g., DNA sequencing, RNA sequencing). Such a polynucleotide sequencer may receive a sample as input, perform the steps of decomposing and analyzing the sample, and generate read data representing the sequence of the read fragments of (multiple) polynucleotides in the sample. The polynucleotide sequencer may fragment and / or combine the DNA (or some other polynucleotides) from the sample to generate a set of polynucleotide fragments. The fragments may then be amplified (e.g., using polymerase chain reaction (PCR)) to generate a copy of each polynucleotide fragment. The polynucleotide sequencer may then sequence the amplified polynucleotide fragments using, for example, imaging techniques that illuminate the fragment and measure the light reflected from it to determine the nucleotide sequence of the fragment. These nucleotide sequence reads may then be output as read data (e.g., a text file with nucleotide sequence and other metadata) and stored on a storage medium.

[0061] Read data alignment can include any combination of hardware and software that receives raw polynucleotide fragment read data and generates aligned read data. In some embodiments, the read data is aligned to a reference genome (although one or more nucleotides or nucleotide fragments within the read fragment may be different from the reference genome). In some cases, a polynucleotide sequencer can also align the read fragments and output aligned read data.

[0062] The aligned read data can be any signal or data indicating the read data and the way each fragment in the read data is aligned. An example data format of the aligned read data is the SAM format. SAM files are tab-delimited text files including sequence alignment data and related metadata. Other data formats (e.g., stacked formats) can also be used.

[0063] The variant calling method / system can be any combination of hardware and software that detects variants in aligned read data and outputs potential variants. The variant caller can identify nucleotide variations (e.g., heterozygous SNPs) between multiple aligned reads at a specific position on a gene, identify nucleotide variations (e.g., homozygous SNPs) between one or more aligned reads at a specific position on a gene and a reference genome, and / or detect any other type of variation within the aligned read data. The variant caller can output data indicating the detected variants in various file formats, such as a variant call format (VCF) that specifies the location of the variant (e.g., chromosome and position), the type of the variant, and other metadata.

[0064] B. Reference genome

[0065] As described herein, "reference genome" can refer to the polynucleotide sequencing data and / or the relevant predetermined nucleotide sequence of a particular sample. This may include a DNA sequence (e.g., for the genome of plants, animals, bacteria, DNA viruses, etc.), an RNA sequence (e.g., for the genome of RNA viruses), or some other polynucleotide sequences of an organism of interest. The reference genome can also include information about the sample, such as its biopsy source, sex, species, phenotypic data and other characteristics. The reference genome can also be referred to as a "gold standard" or "platinum" genome, indicating a high confidence level in the accuracy of the determined nucleotide sequence. An exemplary reference genome is NA12878 sample data and genome. In an example where a sample comprises synthetic DNA or other synthetic polynucleotides (e.g., a sample comprising information in a sequence or other characteristics of a synthetic DNA), the reference genome can be a record of a baseline, unmodified or other reference state of the synthetic DNA in the sample.

[0066] C. Variant Types and Detection

[0067] As described herein, a genome may include multiple chromosomes, each of which may include genes. Each gene may be present in a position on a chromosome that is referred to as a "gene locus". The difference between genes in different samples (i.e., one or more variants at a specific gene locus) may be referred to as alleles. In short, a specific set of alleles in a sample may form the "genotype" of the sample.

[0068] Two genes, or more generally, any nucleotide sequence that differs from one another (in length, nucleotide bases, etc.), may include one or more variants. In some cases, a single sample may contain two different alleles at a particular gene locus; such variants may be referred to as "heterozygous" variants. Heterozygous variants may exist when a sample inherits one allele from one parent and a different allele from the other parent; since diploid organisms (e.g., humans) inherit a copy of the same chromosome from each parent, variation may exist between the two chromosomes. In other cases, a single sample may contain genes that differ from a reference genome; such variants may be referred to as "homozygous" variants.

[0069] Many different types of variants may exist between two different alleles. When two genes have different nucleotide bases at a specific position of the gene, there is a single nucleotide polymorphism (SNP) variant. When a gene comprises a nucleotide sequence, and another gene comprises a part of the nucleotide sequence (removing one or more nucleotide bases) and / or comprises other nucleotide bases (insertion), there is an insertion or deletion (INDEL) between the two genes. There may also be structural differences between the two genes, such as repetitions, inversions, and copy number variations (CNVs).

[0070] Depending on the sensitivity and implementation of the variant caller, read data from the entire genome may include millions of potential variants. Some of these potential variants may be true variants (such as those described above), while others may be false positive detections.

[0071] III. Exemplary Polynucleotide Barcode Insertion and Sequencing

[0072] In various applications, it is desirable to clearly determine the sequence of DNA, RNA or some other target polynucleotide in a sample. However, the cost and capacity limitations of current sequencing technologies, as well as the limitations of the properties of the target polynucleotide of interest and the sample comprising the target polynucleotide, often result in fragmented or unclear read lengths, from which it is difficult to determine the "true" sequence of the target polynucleotide. The systems and methods provided herein improve the sequencing process by introducing polynucleotide barcodes into the target polynucleotides before sequencing. The presence of the inserted barcodes can then be used to improve fragment alignment, variant detection or other aspects of sequencing, thereby resulting in a reduction in the ambiguity of the target polynucleotides and / or a group of target polynucleotides in the sample or otherwise improved sequencing.

[0073] These difficulties may be related to the loss of "distal information" about the relative positions of sequenced fragments in the target polynucleotide. This information loss can be compensated in a variety of ways, for example, by inference based on overlaps or other relationships between different sequence fragments and / or between sequence fragments and known good reference sequences. However, even with the benefit of this, it is not possible to unambiguously determine the sequence of the target polynucleotide.

[0074] For example, if the target polynucleotide includes a repeat-containing region (e.g., a region including an unknown number of trinucleotide repeat sequences, tandem repeats, or other repetitive subsequences), and at least one single sequence fragment of the available fragments does not completely span the repeat region, it is not possible to completely and unambiguously determine the number of repeats present in the repeat region. Determining the number / length of repeats in the repeat region of a target polynucleotide may be particularly relevant in practical applications of sequencing, for example, in diagnosing and / or studying trinucleotide repeat disorders or other phenomena associated with the presence, length, or other properties of the repeat-containing region of a target polynucleotide.

[0075] This ambiguity is expressed through Figure 1A , which shows a first fragment polynucleotide sequence 100a and a second fragment polynucleotide sequence 100b that have been read from a sample and include a repeat sequence 105 (in this example, a trinucleotide repeat, although other types of tandem repeats or other repeat structures are also possible). The identities of the bases in the sequences 100a, 100b are represented by the filled textures of the squares in the sequence, each square representing the corresponding nucleobase of the polynucleotide sequence. Therefore, when the target polynucleotide is DNA, different textures can represent adenine, thymine, guanine and cytosine. When the target polynucleotide is RNA, different textures can represent adenine, uracil, guanine and cytosine. When the target polynucleotide is some other kind of polynucleotide (e.g., an artificial polynucleotide composed of artificial and / or natural nucleobases, a hybrid DNA / RNA polynucleotide), different textures can represent the various nucleobases present in the target polynucleotide.

[0076] The respective ends of the first sequence segment 100a and the second sequence segment 100b each terminate with multiple instances of the repeat sequence 105. Therefore, in the absence of additional information about the relative positions of the segments 100a, 100b along the underlying target polynucleotide (e.g., additional sequence segments that completely span the repeat-containing region), there is insufficient information to unambiguously determine the sequence of the target polynucleotide. For example, Figure 1B A first possible target polynucleotide sequence 100c is shown having a repeat-containing region 107 including three instances of a repeat sequence 105. Based on the information present in the first sequence segment 100a and the second sequence segment 100b, this is the shortest possible length of the repeat region of the underlying real target polynucleotide sequence. The repeat region can be longer, for example, can contain 9 or more instances of the repeat sequence 105, such as Figure 1C A second possible target polynucleotide sequence 100d is shown as containing a repeat region 109 .

[0077] Figure 1A-Figure 1CThe repeat number / length ambiguity shown in is only a related ambiguity that can be generated by sequencing a sample using current sequencing methods and can be fully or partially alleviated by applying the systems and methods disclosed herein. For example, the difficulty of sequencing a target polynucleotide in a sample may be related to the presence of many different polynucleotides in the sample. In such examples, it is not only difficult to align the various fragment read sequences, but also difficult to group them correctly together. Therefore, it may be difficult to determine, for example, whether an individual is heterozygous at a specific position, or conversely, whether a single nucleotide polymorphism (SNP) or other variant observed in a specific fragment read sequence represents an error in the sequencing process. This problem may be exacerbated by the lack of a good reference genome and / or the lack of robust statistical information about variants or other sequence information of specific genes, chromosomes or other gene loci within the target polynucleotide. In another example, a plurality of different polynucleotides in a sample may include a plurality of different isoforms of a specific RNA. In such examples, it may be difficult to unambiguously determine the sequence of all the different isoforms of RNA in a sample due to, for example, difficulties in associating fragment read sequences with the correct isoform, difficulty in assessing whether an observed variant represents a true variant in one of the isoforms, an error in the sequencing process, or due to other factors.

[0078] The systems and methods described herein solve these and other problems in sequencing by inserting a number of "barcode" polynucleotide sequences into target polynucleotides before sequencing fragments of the target polynucleotides, etc. The barcode sequence can then be used to determine the sequence of one or more target polynucleotides in an improved manner. Such a process may include using the barcode sequence present in the fragment read sequence to identify the fragment read sequence as belonging to the same target polynucleotide (e.g., the same one in a pair of chromosomes, the same isoform of multiple isoforms of RNA translated from the same gene), to align the fragment read sequences (e.g., to eliminate the ambiguity about the presence of indels, the number of repeat sequences, or some other ambiguity that would exist in the absence of barcode sequences), and / or to facilitate and / or improve some other aspects of sequencing. Alternatively, existing alignment or other sequencing algorithms (after appropriate modification) can be applied to align the fragment read sequences from samples into which such barcodes have been inserted. The benefit of such examples is that the presence of the barcode may break up repetitive sequences or otherwise add a certain amount of "randomness" that can improve alignment or other sequence generation processes, with the barcode later removed from the generated sequences.

[0079] Figure 2A-2E An exemplary process is shown that includes inserting a polynucleotide barcode into a target polynucleotide to generate a sequence of the target polynucleotide. Figure 2ATarget polynucleotide 200 is shown. Target polynucleotide 200 can be composed of DNA, RNA, some other natural or artificial nucleobases and / or some combinations thereof. For example, target polynucleotide 200 can be cDNA generated from RNA of interest. Target polynucleotide 200 can be the entirety of a chromosome (e.g., a specific chromosome in a pair of chromosomes), mRNA (e.g., a specific isoform of mRNA transcribed from a specific gene locus or gene), or other naturally terminated polynucleotides, or can be a specific portion thereof, for example, a specific gene, a group of genes, alleles, or other specific gene loci within a larger polynucleotide.

[0080] The target polynucleotide 200 can be isolated and / or purified so that it is the only polynucleotide present in the sample. Alternatively, the target polynucleotide 200 can be one of a plurality of different polynucleotides present in the sample (e.g., other chromosomes or fragments thereof, other isoforms of RNA corresponding to the same gene locus or gene). The target polynucleotide 200 can be amplified (e.g., via a polymerase chain reaction (PCR) process or some other amplification process), fragmented (e.g., by applying a restriction enzyme), combined and / or processed in some other manner.

[0081] Figure 2B A set of polynucleotide barcodes 210 that can be inserted into a target polynucleotide 200 is shown. The polynucleotide barcode 210 can have a specified length to facilitate detection of the barcode in a read sequence of a fragment of the target polynucleotide 200. The polynucleotide barcode 210 can include a library of different barcodes. In some examples, the polynucleotide barcode 210 can have a sequence that is not present anywhere in the target polynucleotide 200 (e.g., does not match any subsequence within the reference genome of the target polynucleotide 200) to facilitate detection of the barcode in the read sequence of a fragment of the target polynucleotide 200. A set of polynucleotide barcodes 210 can include multiple copies of a single barcode sequence to reduce costs. In such examples, the correspondence between the occurrences of a specific barcode sequence in different fragment read sequences can be determined by comparing the read sequences around the specific barcode sequence. If the read sequences around the specific barcode sequence in two different fragment read sequences do not match, they are unlikely to represent the same instance of the inserted polynucleotide barcode.

[0082] The set of polynucleotide barcodes 210 can be inserted into the target polynucleotide 200. This can be accomplished by introducing a probe comprising a polynucleotide barcode 210 into a sample comprising the target polynucleotide 200 and / or a fragment or copy thereof. Such a probe can include an insertion vector (e.g., CRISPR-Cas9, Tn5 transposase) to insert the specific polynucleotide barcode of the probe into the target polynucleotide 200. The probe may include other elements or features. In some examples, the probe can be configured to insert the barcode into a specified position in the target polynucleotide 200 (e.g., to facilitate sequencing of a specific gene locus within the target polynucleotide 200, to increase the likelihood of inserting the barcode into a repetitive region or other region that is prone to false variant calls or other ambiguous sequencing phenomena). In some examples, the probe can be inserted into a payload polynucleotide, which includes a polynucleotide barcode and some additional polynucleotide sequences, such as forward and / or reverse primer sequences, one or more additional barcodes, an end cap polynucleotide sequence that facilitates the insertion of the remainder of the payload polynucleotide into the target polynucleotide 200 via an insertion vector, and the like.

[0083] Figure 2C A post-insertion polynucleotide 220 is shown, which is a target polynucleotide 200 into which a plurality of polynucleotide barcodes 210 are inserted. As shown, the post-insertion polynucleotide 220 has a single instance of each unique polynucleotide barcode inserted therein. However, in practice, a single target polynucleotide may have multiple copies of each barcode in a set of polynucleotide barcodes inserted therein. In addition, in some examples, multiple copies, fragments, and / or copies of fragments of the target polynucleotide 200 may be present in the sample, in which case each of the copies, fragments, and / or copies of fragments may have a correspondingly different pattern of polynucleotide barcodes inserted therein. Inserting polynucleotide barcodes into multiple copies, fragments, and / or copies of fragments in a target polynucleotide may have the benefit of allowing a larger proportion of target polynucleotides to be clearly sequenced at a lower barcode insertion rate.

[0084] The post-insertion polynucleotide 220 can then be sequenced to generate multiple reads of the target polynucleotide 200 in the form of multiple reads 230 of the post-insertion polynucleotide 220, such as Figure 2DAs shown. This can include applying various next-generation sequencing technologies or other methods to generate read lengths of portions of the inserted polynucleotide 220. The sequencing process can include, after inserting the barcode, amplifying the inserted polynucleotide 220, fragmenting the inserted polynucleotide 220, amplifying fragments of the inserted polynucleotide 220 after such fragmentation, or applying some other process to sequence the inserted polynucleotide 220. Sequencing the inserted polynucleotide 220 to generate multiple read lengths 230 can include fragmenting the inserted polynucleotide 220 and then sequencing the fragments. Additionally or alternatively, the read length can start from a specified position (e.g., a primer sequence) on the inserted polynucleotide 220 and / or its fragments. In some examples, such a primer can be inserted together with a barcode, for example, to ensure that each read length 230 starts with a barcode sequence.

[0085] like Figure 2D As shown, each read in the plurality of reads 230 includes at least one barcode. Furthermore, the specific pattern of the presence of the barcodes represented in the plurality of reads 230 means that the plurality of reads 230 can be stitched together based on the barcodes without ambiguity regarding the ordering or degree of overlap of the reads. The output of this alignment process is Figure 2E A preliminary sequence 240 is shown. The sequence of the target polynucleotide 200 can then be recovered by removing the barcode sequence from the preliminary sequence 240.

[0086] Inserting a polynucleotide barcode into a target polynucleotide can facilitate unambiguous recovery of the sequence of the target polynucleotide in a variety of ways, even in the face of sequence repeats, indels, or other features in the target polynucleotide. In a first example, Figure 3 Portions of a first read segment 310a and a second read segment 320a are shown. A first polynucleotide barcode 300a is present in the first read segment 310a and the second read segment 320a, and a second polynucleotide barcode 300b is present in the second read segment 320a. The target polynucleotide into which the barcodes 300a, 300b are inserted and from which the read segments 310a, 320a are read includes multiple repeats (trinucleotide repeats, although tandem repeats or other repeat sequence structures are also possible) of a repetitive sequence 305. The first polynucleotide barcode 300a is inserted within the repeat region between instances of the repetitive sequence 305. Therefore, the number of instances of the repetitive sequence 305 can be unambiguously determined, which would not be possible without the insertion of the barcodes 300a, 300b (e.g., in combination with Figure 1A-Figure 1C In another example, an indel may be present (or absent) between barcode sequences inserted into a target polynucleotide, and the inserted barcode may help to unambiguously determine the presence (or absence) of the position of the indel in the target polynucleotide.

[0087] Properties of the sequencing process (e.g., properties related to the average length of the read fragments) and / or properties of the barcode insertion process (e.g., properties that increase the density of barcode insertions) can be specified to increase the likelihood that all or significantly contiguous portions of the target polynucleotide 200 can be unambiguously sequenced in this manner. In practice, however, there may be some portions of the target polynucleotide that are not represented by read fragments that contain barcodes sufficient to completely and unambiguously recover the underlying sequence of the target polynucleotide. For example, a particular portion of the target polynucleotide may not be represented by read fragments that also contain barcodes on either side of the particular portion (those barcodes are also represented in other read fragments, etc.), or may not be represented in any read fragments at all. In this case, conventional read alignment methods (between different read fragments, or between read fragments and a reference genome) can be applied to recover the sequence of the particular portion of the target polynucleotide.

[0088] To increase the likelihood of unambiguously sequencing the entirety of the target polynucleotide sequence, the target polynucleotide can be amplified prior to inserting the barcodes. Thus, multiple different copies of the target polynucleotide will insert different sets of barcodes at different locations within the target polynucleotide. Thus, different long contiguous segments of different copies can be unambiguously recovered. These long contiguous sequences can then be aligned to each other to generate a higher quality, more accurate sequence for the underlying target polynucleotide.

[0089] Polynucleotide barcodes can be specified and / or manufactured in a variety of ways. The barcodes can have a length that facilitates detection of the barcode in the read fragment (thereby allowing the detected barcode to be used to generate the sequence of the target polynucleotide). For example, the polynucleotide barcode can be tens or hundreds of nucleotides long. A variety of methods can be used to detect and identify barcodes in the read fragment so that multiple copies of a single polynucleotide barcode can be inserted into a single target polynucleotide.

[0090] This can include identifying instances of a particular polynucleotide barcode in a plurality of different read fragments, and then using the neighborhood of the identified barcodes to determine correspondence between the read fragments. Figure 4A An exemplary first read segment 410a, a second read segment 420a, and a third read segment 420c are shown in which copies of the polynucleotide barcode 400 are located. The position of the polynucleotide barcode 400 in each read segment 410a, 420a, 430a can be determined (e.g., by a pattern matching process). Then, the nucleotides in the neighborhood of each copy of the barcode 400 can be used to identify whether each identified copy of the barcode 400 corresponds to the same insertion instance of the polynucleotide barcode 400 and should therefore be aligned. Figure 4AIn the example shown, such a process would result in an association of the first read segment 410a and the second read segment 420a, but not the third read segment 430a, because the neighborhoods of the first read segment 410a and the second read segment 420a around the barcode 400 match each other, but do not match the neighborhood of the third read segment 430a around the barcode 400.

[0091] As described above, detecting a barcode sequence within a read and / or a preliminary sequence (e.g., 240) to remove an inserted barcode sequence can include using pattern matching or other techniques to detect the presence of a sequence that matches the sequence of the barcode within the read or preliminary sequence. When a reference genome is available for all or a portion of the target polynucleotide, the read and / or preliminary sequence can be compared to the reference genome to detect the presence of the barcode sequence. This is accomplished by Figure 4B 4 is shown, which shows a read segment 420b into which a polynucleotide barcode 400 has been inserted. A portion of a reference genome 410b corresponding to the read segment 420b is also shown. The read segment 420b can be compared to the reference genome 410b to detect the polynucleotide barcode 400 in the read segment 420b by detecting the presence of the sequence of the polynucleotide barcode 400 in the read segment 420b and / or by detecting the presence of a sequence (barcode) in the read segment 420b inserted into the sequence of the reference genome 410b. Such a process can be performed as an alternative to pattern matching or other methods to detect barcode sequences in read segments and / or to enhance such a process.

[0092] The specific nucleotide sequence represented by a single polynucleotide barcode in a library of such barcodes can be randomly selected, for example, generated as a result of randomly generated oligonucleotides. Alternatively, as described herein, the sequence of the barcode can be selected to enhance certain aspects of its use in sequencing the target polynucleotide. For example, in the case where a reference genome can be used for the target polynucleotide, the sequence of the polynucleotide barcode can be selected as a sequence that does not occur and / or is unlikely to occur despite the presence of a threshold level of copy errors relative to the reference genome. Once the sequence of the barcode is determined, the corresponding polynucleotide barcode can be generated using, for example, available methods for synthesizing oligonucleotides having a specific sequence.

[0093] Once the barcodes are generated, they can be amplified, conjugated to an insertion vector or otherwise incorporated into a probe, or otherwise processed to provide a library of polynucleotide barcodes that can be inserted into a target polynucleotide to facilitate sequencing of the target polynucleotide. In some examples, the polynucleotide barcodes themselves can be sequenced to determine and / or confirm the sequence of the polynucleotide barcode (e.g., in examples where the polynucleotide barcodes are generated by a random or semi-random synthesis process).

[0094] As described above, the polynucleotide barcode can be inserted into the target polynucleotide after amplification, fragmentation or some other process of the target polynucleotide. In some examples, the polynucleotide barcode can be inserted into the target polynucleotide, a fragment of the target polynucleotide and / or inserted into the target polynucleotide or its fragment multiple times, interspersed with, for example, additional amplification, fragmentation and / or other sample preparation processes. Such a process can be performed, for example, to further improve the "randomness" in the read fragment introduced by inserting the polynucleotide barcode, and further improve the quality and / or accuracy of the sequence of the target polynucleotide determined thereby. In this repeated insertion process, the sample contents can be sequenced at multiple different points. For example, multiple polynucleotide barcodes can be inserted into the target polynucleotide, and multiple read fragments can be sequenced therefrom (for example, after amplification). Then, additional multiple polynucleotide barcodes can be inserted into the target polynucleotide (and / or its copy, fragment and / or copy of the fragment), and multiple additional read fragments can be sequenced from the double inserted sample.

[0095] IV. Exemplary Probes

[0096] The systems and methods described herein include inserting a polynucleotide barcode into a target polynucleotide to facilitate and / or improve sequencing of a target polynucleotide and / or a selected portion thereof. A set of polynucleotide barcodes can be inserted into a target polynucleotide using a variety of methods. In some examples, this can include creating a plurality of probes, each of which includes an insertion vector and a payload polynucleotide (which includes a polynucleotide barcode). An insertion vector is a structure (e.g., protein, DNA, RNA, and / or other substance or structure) configured to insert a payload polynucleotide into another polynucleotide (e.g., into a target polynucleotide).

[0097] Insertion vector can include CRISPR-Cas9, CRISPR-Cas12a, CRISPR related to some other proteins or protein complexes, Tn5 transposase, Tn7 transposase, some other transposases or some other insertion vectors that can play the role of inserting the payload polynucleotide containing barcode into the target polynucleotide. Insertion vector can insert payload polynucleotide into the target polynucleotide in random position and / or the specified position in the target polynucleotide (for example, the specified position complementary to the guide RNA (Guide RNA, gRNA) of the insertion vector in the target polynucleotide). If the insertion vector is configured to insert the payload into the specified (multiple) position, the (multiple) position can be designated as the target position of particular interest in the target polynucleotide, for example, near the position of SNP, trinucleotide repeat, indel or other variants related to a specific disease or disorder.

[0098] The payload polynucleotide of the probe includes at least one polynucleotide barcode, but may include additional elements. For example, the payload polynucleotide may include forward and / or reverse primer sequences. This may be performed by providing a primer sequence to facilitate the generation of a read fragment including at least one polynucleotide barcode, which initiates the amplification of a target polynucleotide having a barcode inserted at a position close to the barcode of the payload polynucleotide. Additionally or alternatively, the payload polynucleotide may include a restriction site sequence or other sequence to facilitate the targeted cutting of the polynucleotide after the insertion of the payload polynucleotide. These payload contents may be included to facilitate the generation of a read fragment including at least one polynucleotide barcode near the end of the fragment by facilitating the fragmentation of the target polynucleotide close to the barcode of the payload polynucleotide, for example, to facilitate the generation of a read fragment starting and ending with an inserted polynucleotide barcode, or otherwise including the generation of a read fragment of an inserted polynucleotide barcode close to both ends of the read fragment. In some examples, the payload polynucleotide may include a sequence that facilitates the insertion of an insertion vector associated with and / or by an insertion vector. Depending on the details of the insertion vector, these sequences may or may not be inserted into the target polynucleotide. The payload polynucleotide may include multiple polynucleotide barcodes, for example, to facilitate the reuse of the barcode or to provide some other benefits. In the example where the payload polynucleotide includes a sequence (e.g., a primer sequence, a restriction site, or other cutting-targeting sequence) other than an explicit "barcode" sequence, this additional sequence may be considered to be a part of the barcode and / or may be identified together with the barcode sequence to detect the presence of the barcode sequence in the read fragment. In some examples, the payload may include a "gap sequence" (e.g., placed between the forward primer sequence and the reverse primer sequence) flanked by any of the above-mentioned features to provide a targeted cutting site (e.g., for a restriction enzyme), a non-critical base that allows the variability of the degree or position of the polynucleotide cutting / insertion / etc., or to provide some other benefits.

[0099] V. Exemplary Dual-Linked Probes and Related Methods

[0100] As described above, inserting a polynucleotide barcode into a target polynucleotide can provide a variety of benefits for determining the sequence of a target polynucleotide. In addition to a single barcode sequence, these methods can also be expanded by inserting additional content into the target polynucleotide. For example, forward and / or reverse primer sequences can be inserted so that all (or increased amounts) of read fragments generated from the target polynucleotide into which a barcode is inserted start with a barcode sequence. Doing so can provide many benefits, for example, for increasing the likelihood that a read fragment includes two or more inserted barcode sequences, for allowing targeting of the outermost amplicon of interest close to these inserted primer sequences, and / or for facilitating clear sequencing of a portion of a target polynucleotide. Additionally or alternatively, the insertion of a primer together with a polynucleotide barcode can allow a polynucleotide barcode to be repeatedly inserted into a target polynucleotide without segmentation, even when using a sample containing very few copies of a target polynucleotide. This may have the benefit of allowing read length multiplexing, or otherwise improve the accuracy / quality of sequencing processes and / or sequence data generated thereby.

[0101] In a specific example, a forward primer sequence and a reverse primer sequence can be inserted together with one or more associated barcode sequences. Subsequently, the forward primer sequence and the reverse primer sequence can be used to amplify and read a fragment of the target polynucleotide starting with the inserted barcode(s), so that the association between two different inserted barcodes, or the association between an inserted barcode at the beginning of two different read fragments and its reverse direction can be used to align the ends of the two different read fragments.

[0102] An example of such a "double-linked" polynucleotide barcode payload sequence 500a that can be inserted into a target polynucleotide is Figure 5AAs shown. Payload sequence 500a includes a first barcode ("AAAA") and a second barcode ("BBBB"). The first barcode and the second barcode can be the same or different sequences (for example, one sequence is opposite to the other sequence). Payload sequence 500a also includes a forward primer sequence (indicated by an arrow facing the right) and a reverse primer sequence (indicated by an arrow facing the left). When inserted into a target polynucleotide, the forward primer sequence and the reverse primer sequence can cause a transcriptase or other substance to start copying the target polynucleotide into which the payload is inserted at the forward primer sequence or the reverse primer sequence, respectively, in the forward or reverse direction of the target polynucleotide. Therefore, the copy fragment of the target polynucleotide starting from the forward primer sequence or the reverse primer sequence will start with the second ("BBBB") or the first ("AAAA") barcode sequence, respectively. If these barcode sequences are sequenced, these barcode sequences can be detected in the copy fragments. In response to such detection, the correspondence between the forward second barcode and the reverse first barcode can be determined and used to align the sequences of the copied fragments together.

[0103] Alternatively, the primer sequences can be opposite to each other, allowing the use of a single barcode sequence (which will be reverse sequenced on the fragment copied from the reverse primer). An example of such a polynucleotide barcode payload sequence 500b that can be inserted into the target polynucleotide is as follows: Figure 5B As shown. Payload sequence 500b includes a barcode ("AAAA"), flanked by a forward primer sequence (indicated by an arrow facing the right) and a reverse primer sequence (indicated by an arrow facing the left). When inserted into a target polynucleotide, the forward primer sequence and the reverse primer sequence can cause a transcriptase or other substance to start copying the target polynucleotide into which the payload has been inserted at the forward primer sequence or the reverse primer sequence, respectively, in the forward or reverse direction of the target polynucleotide. Therefore, the copy fragment of the target polynucleotide starting from the forward primer sequence or the reverse primer sequence will start with the barcode sequence or the reverse barcode sequence, respectively. Repeated size selection of the copy fragment can be used to exclude copy fragments consisting only of a barcode and a minimal flanking sequence. If the copy fragment is sequenced, the barcode sequence can be detected in the copy fragment. In response to this detection, the correspondence between the barcode and its reverse sequence can be determined and used to align the sequences of the copy fragments together.

[0104] Figure 6The generation and alignment of such fragments is shown. The target polynucleotide 600 has multiple double-linked polynucleotide barcodes inserted therein (indicated by the double-color subsequence along the length of the target polynucleotide 600), including a first specific double-linked polynucleotide barcode 615, which includes a first barcode 615a and a second barcode 615b. The inserted double-linked polynucleotide barcodes additionally include a second specific double-linked polynucleotide barcode 617 (which includes a third barcode 617a and a fourth barcode 617b), and a third specific double-linked polynucleotide barcode 619 (which includes a fifth barcode 619a and a sixth barcode 619b). The first read fragment 610a is generated by transcribing the target polynucleotide 600 from the forward primer sequence of the first specific double-linked polynucleotide barcode 615, from the reverse primer sequence of the third specific double-linked polynucleotide barcode 619, and / or by fragmenting the target polynucleotide 600 between the barcodes 615a, 615b and 619a, 619b of the first specific double-linked polynucleotide barcode 615 and the third specific double-linked polynucleotide barcode 619, respectively, and transcribing the fragments and then sequencing the transcribed polynucleotides. Such sequencing can include paired end sequencing of the fragments, amplifying the fragments and sequencing the amplified fragment copies from the first and / or second ends of the fragments, or using some other sequencing technology. Therefore, the first barcode 615a is near one end of the first read fragment 610a, and the sixth barcode 619b is near the other end of the first read fragment 610a. The second read fragment 610b is generated by transcribing the target polynucleotide 600 from the reverse primer sequence of the first specific double-linked polynucleotide barcode 615, from the reverse primer sequence of the second specific double-linked polynucleotide barcode 617, and / or by fragmenting the target polynucleotide 600 between the barcodes 615a, 615b and 617a, 617b of the first specific double-linked polynucleotide barcode 615 and the second specific double-linked polynucleotide barcode 617, respectively, transcribing the fragments and then sequencing the transcribed polynucleotides. Thus, the second barcode 615b is near one end of the second read fragment 610b, and the third barcode 617a is near the other end of the second read fragment 610b. The correspondence between the first barcode 615a and the second barcode 615b can be determined and used to align (or "stitch together") the first read fragment 610a and the second read fragment 610b, such as Figure 7 The sequences are stitched together as shown in 620.

[0105] Note that this is only a single example of a situation in which inserting such a dual-linked barcode into a target polynucleotide can result in lower cost, faster, more accurate or otherwise improved sequencing of the target polynucleotide. In practice, amplicons are likely to begin and / or end with one barcode in a pair and are unlikely to extend to extend across and beyond adjacent barcode insertions (particularly in instances where restriction enzymes or other means have been used to fragment the target polynucleotide between the paired barcodes of each of the inserted dual-linked polynucleotide barcodes), adding information about the links between different reads represented in each read or in a set of reads (the portion of the read fragment sequence extending through and beyond the dual-linked barcode insertion is unlikely to contain any additional information when compared to another fragment read sequence starting from one of the dual-linked barcode insertion barcodes). In addition, additional restriction sequences or other cutting-targeting sequences in the dual-linked barcode payload between barcodes allow the target polynucleotide (or its copies) to be cut into fragments of similar size, resulting in more uniform amplification of the target polynucleotide, particularly in situations where the available amount of input target polynucleotides is low.

[0106] Multiple stitching processes can be performed based on linked polynucleotide barcodes detected in additional read fragments to generate a stitched-together sequence that spans all or a significant contiguous portion of the target polynucleotide 600. The barcodes and associated inserted polynucleotide sequences (e.g., restriction site sequences, primer sequences, sequences that connect other sequences together, partial sequences of the insertion vector used to insert the barcode and other sequences, mosaic end sequences used as sacrificial sequences to ensure that the insertion vector inserts all of the barcode or other payload sequence into the target polynucleotide) can then be removed to provide the sequence of the target polynucleotide prior to insertion of the barcode.

[0107] The benefit of inserting a primer sequence together with a barcode sequence is that the primer sequence can direct the transcription and amplification of a fragment of the target polynucleotide into which the barcode is inserted, without binding to the target polynucleotide or otherwise fragmenting the target polynucleotide. Thus, the polynucleotide barcode can be inserted into the target polynucleotide in several rounds, generating a read fragment of the target polynucleotide after each insertion. This can provide richer read fragment data, improving the quality and / or accuracy of the resulting sequence.

[0108] The payload polynucleotide sequence described herein (e.g., including one or more barcode sequences, primer sequences, restriction site sequences, and / or some other sequences) can be generated in a variety of ways. In some examples, the sequence of the payload polynucleotide can be fully specified, and then the payload polynucleotide is generated by an oligonucleotide synthesis process. However, this practice may be extremely expensive. Therefore, a variety of alternative methods can be used to generate a library of double-linked barcode payload polynucleotide sequences. For example, existing barcode sequences and primer sequences can be combined to form payload polynucleotides. Existing barcode polynucleotides can be generated via directed or random oligonucleotide synthesis. In the case of using some aspects of randomness to generate payload polynucleotides, the payload polynucleotide can be sequenced after synthesis to determine the association between the barcodes of each payload polynucleotide.

[0109] Optionally or alternatively, the complement of a polynucleotide comprising a single barcode and primer sequence can be grown on the polynucleotide to generate its complement. A hairpin can then be added to denature and separate the complementary strands of the polynucleotide to provide a double-linked barcode polynucleotide comprising a forward primer, a reverse primer, and two barcodes that are reverse complements of each other.

[0110] This process is Figure 8A-8D Shown in. Fig. 8A A template polynucleotide including a first barcode polynucleotide ("AAAA") and a primer sequence (arrow toward the left) is shown. Figure 8B The formation of a complementary polynucleotide on the template polynucleotide (eg, via a polymerase chain reaction) is shown. Thus, the complementary polynucleotide includes a second barcode polynucleotide ("AAAA") that is complementary to the first barcode polynucleotide and a second primer sequence. Figure 8C The addition of a hairpin 800 is shown to connect the template polynucleotide to the complementary polynucleotide. Fig.8D The double-linked barcode polynucleotide is shown after denaturation to separate the template polynucleotide and the complementary polynucleotide. Thus, the double-linked barcode polynucleotide includes a first barcode polynucleotide and a second barcode polynucleotide, the second barcode polynucleotide being a reverse complementary version of the first barcode polynucleotide.

[0111] VI. Exemplary Methods

[0112] Fig. 9An exemplary method 900 is depicted. Method 900 includes adding a probe to a sample comprising a target polynucleotide, wherein the probe includes a payload polynucleotide and an insertion vector, wherein the payload polynucleotide includes a first polynucleotide barcode, a reverse primer, a forward primer, and a second polynucleotide barcode, and wherein the insertion vector inserts the payload polynucleotide into the target polynucleotide (910). Method 900 additionally includes administering an amplification agent to generate: (i) a first copy of the target polynucleotide with the payload polynucleotide inserted therein, starting with the reverse primer, such that the first copy includes a complement of the first polynucleotide barcode near an end of the first copy, and (ii) a second copy of the target polynucleotide with the payload polynucleotide inserted therein, starting with the forward primer, such that the second copy includes a complement of the second polynucleotide barcode near an end of the second copy (920). Method 900 may include additional steps or features.

[0113] Fig.10 An exemplary method 1000 for generating a payload polynucleotide is depicted. The method 1000 includes obtaining a template polynucleotide including a first polynucleotide barcode and a first amplification primer (1010). The method 1000 further includes forming a complementary polynucleotide on the template polynucleotide such that the complementary polynucleotide includes a second amplification primer and a second polynucleotide barcode (1020). The method 1000 further includes forming a hairpin between the template polynucleotide and the complementary polynucleotide (1030). The method 1000 further includes dissociating the complementary polynucleotide from the template polynucleotide via a denaturation process (1040). The method 1000 may include additional steps or features.

[0114] Fig.11 An exemplary method 1100 is depicted. The method 1100 includes inserting a plurality of polynucleotide barcodes into a target polynucleotide contained within a sample, wherein the plurality of inserted polynucleotide barcodes include a first polynucleotide barcode and a second polynucleotide barcode (1110). The method 1100 further includes, after inserting the plurality of polynucleotide barcodes into the target polynucleotide, sequencing at least a portion of the sample multiple times to obtain a plurality of reads of the target polynucleotide, wherein a first read in the plurality of reads includes the first polynucleotide barcode, and wherein a second read in the plurality of reads includes the first polynucleotide barcode and the second polynucleotide barcode (1120). The method 1100 further includes determining a sequence of the target polynucleotide based on the plurality of reads of the target polynucleotide (1130).

[0115] Determining the sequence of the target polynucleotide (1130) includes: determining a preliminary sequence of the target polynucleotide, wherein determining the preliminary sequence includes stitching the first read and the second read together so that the first polynucleotide barcode in each of the first read and the second read overlaps (1132). Determining the sequence of the target polynucleotide (1130) also includes: removing the sequences of the first polynucleotide barcode and the second polynucleotide barcode from the preliminary sequence (1134).

[0116] Method 1100 may include additional steps or features.

[0117] It should be understood that the arrangements described herein are for illustrative purposes only. Therefore, those skilled in the art will appreciate that other arrangements and other elements (e.g., machines, interfaces, operations, sequences, and operation groupings, etc.) may be used to replace the elements or arrangements shown or to supplement them.

[0118] VII. Conclusion

[0119] It should be understood that the arrangement described herein is only for illustrative purposes. Therefore, it will be appreciated by those skilled in the art that other arrangements and other elements (e.g., machines, interfaces, operations, sequences, and operation groupings, etc.) may be used alternatively, and some elements may be omitted completely, depending on the desired result. In addition, many of the elements described are functional entities that may be implemented as discrete or distributed components or combined with other components in any suitable combination and position, or may be combined with other structural elements described as independent structures.

[0120] Although various aspects and implementations have been disclosed herein, other aspects and implementations will be apparent to those skilled in the art. The various aspects and implementations disclosed herein are for illustrative purposes only and not for limitation, the true scope being indicated by the appended claims and the full range of equivalents to which these claims are entitled. It should also be understood that the terms used herein are only for the purpose of describing a particular implementation and not for limitation.

Claims

1. A method for polynucleotide barcode insertion, comprising: adding a probe to a sample comprising a target polynucleotide, wherein the probe comprises a payload polynucleotide and an insertion vector, wherein the payload polynucleotide comprises a first polynucleotide barcode, a reverse primer, a forward primer, and a second polynucleotide barcode, and wherein the insertion vector inserts the payload polynucleotide into the target polynucleotide; and administering an amplification agent to generate: (i) a first copy of the target polynucleotide with the payload polynucleotide inserted therein, starting with the reverse primer such that the first copy includes the complement of the first polynucleotide barcode near an end of the first copy, and (ii) a second copy of the target polynucleotide with the payload polynucleotide inserted therein, starting with the forward primer such that the second copy includes the complement of the second polynucleotide barcode near an end of the second copy, in: After adding the probe to the sample, sequencing at least a portion of the sample multiple times to obtain a read length of a first copy of the target polynucleotide and a read length of a second copy of the target polynucleotide; Determining the sequence of the target polynucleotide based on the read length of the first copy and the read length of the second copy, wherein determining the sequence of the target polynucleotide comprises: Determining a preliminary sequence of the target polynucleotide based on the read length of the first copy and the read length of the second copy; and removing the sequences of the first polynucleotide barcode and the second polynucleotide barcode from the preliminary sequence, and in: After administering the amplification agent, inserting an additional plurality of polynucleotide barcodes into the target polynucleotide to obtain additional copies of the target polynucleotide having the additional plurality of polynucleotide barcodes inserted therein; and After inserting the additional multiple polynucleotide barcodes into the target polynucleotide, at least a portion of the sample is sequenced multiple times to obtain reads of additional copies of the target polynucleotide, wherein determining the sequence of the target polynucleotide includes determining the sequence based on the reads of the additional copies of the target polynucleotide.

2. The method according to claim 1, wherein: The insertion vector comprises the Tn5 transposase.

3. The method according to claim 1, wherein: The insertion vector includes CRISPR-Cas9.

4. The method according to claim 1, wherein: Determining the preliminary sequence includes stitching together ends of the first copy and ends of the second copy.

5. The method according to claim 4, further comprising: sequencing the probe to generate a probe sequence; as well as Based on the probe sequence, determining corresponding sequences of the first polynucleotide barcode and the second polynucleotide barcode and determining that the first polynucleotide barcode and the second polynucleotide barcode correspond to each other, wherein stitching the ends of the first copy together with the ends of the second copy is performed in response to determining that the first copy comprises the determined sequence of the first polynucleotide barcode near the end of the first copy and the second copy comprises the determined sequence of the second polynucleotide barcode near the end of the second copy.

6. The method according to any one of claims 1 to 5, further comprising: Prior to adding the probe to the sample, at least a portion of the sample is sequenced multiple times to obtain multiple unmodified reads of the target polynucleotide, wherein removing the first polynucleotide barcode and the second polynucleotide barcode from the preliminary sequence comprises comparing the preliminary sequence to at least one of the multiple unmodified reads of the target polynucleotide.

7. The method according to any one of claims 1 to 5, wherein: The target polynucleotide comprises DNA.

8. The method according to any one of claims 1 to 5, wherein: The target polynucleotide comprises RNA, wherein the target polynucleotide is a first isoform of an RNA sequence, and wherein the sample comprises a second isoform of the RNA sequence, and wherein the first isoform is different from the second isoform.

9. The method according to any one of claims 1 to 5, wherein: The first polynucleotide barcode and the second polynucleotide barcode have the same sequence.

10. The method according to any one of claims 1 to 5, wherein: The second polynucleotide barcode is a reverse complementary version of the first polynucleotide barcode.

11. A non-transitory computer readable medium having stored therein instructions executable by a computing device to cause the computing device to determine a sequence of a target polynucleotide based on sequencing read data generated according to the method of any one of claims 1-10.

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