Design of probes to deplete abundant transcripts

Abstract

Disclosed herein are systems and methods for designing probes for depleting abundant transcripts from a sample. Abundant sequence reads can be determined in a species-agnostic manner, and probes for depleting abundant transcripts can be designed based on the sequence of the most abundant sequence. Also disclosed herein are compositions and kits for depleting abundant transcripts and methods for depleting abundant transcripts.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 950,891, filed on December 19, 2019, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure relates generally to the field of depleting abundant substances, and more particularly to probes designed for depleting abundant substances. Background Art

[0004] One challenge of RNA sequencing for gene expression analysis is that after RNA extraction, most of the extracted material is dominated by a small amount of highly abundant transcripts such as non-coding ribosomal RNA (rRNA). In total RNA samples from human blood, globin messenger RNA (mRNA) can be present at a dominant level. It is necessary to deplete the abundant transcripts in the sample, such as rRNA and mRNA, before RNA sequencing. Summary of the invention

[0005] Disclosed herein are embodiments of systems or methods for designing probes for the abundant sequences of consuming ribonucleic acid transcripts. In some embodiments, the method is controlled by a hardware processor (or a processor, such as a virtual processor) and includes: receiving multiple sequence reads of ribonucleic acid (RNA) transcripts or their products in a sample. The method may include: comparing each of the multiple sequence reads with a reference nucleotide sequence or a subsequence thereof in a plurality of reference nucleotide sequences. The method may include: determining the abundant sequences of a reference nucleotide sequence or a subsequence thereof in a plurality of reference nucleotide sequences. Each of the abundant sequences may have a coverage higher than a coverage threshold. Coverage may be associated with multiple sequence reads aligned with the abundant sequences. The method may include: determining the most abundant sequence with the highest number of coverages in the abundant sequence of the reference nucleotide sequence with a coverage higher than a coverage threshold. The method may include: designing one or more nucleic acid probes for each of the most abundant sequences with the highest number of coverages of the reference nucleotide sequence based on the sequence of the most abundant sequence, the probe length, and the splicing gap.

[0006] In some embodiments, the reference nucleotide sequence in the plurality of reference nucleotide sequences is a reference RNA sequence of a gene. In some embodiments, the reference nucleotide sequence in the plurality of reference nucleotide sequences is a reference deoxyribonucleic acid (DNA) sequence of a gene.

[0007] In some embodiments, the coverage threshold is about 10 to about 10000. In some embodiments, the coverage of an enriched sequence in an enriched sequence is the number of sequence reads aligned to the enriched sequence. In some embodiments, the coverage of an enriched sequence in an enriched sequence is the minimum number of sequence reads aligned to each of a plurality of subsequences of the enriched sequence.

[0008] In some embodiments, one, at least one, or each of the enriched sequences comprises a plurality of contiguous subsequences of a reference nucleotide sequence in a plurality of reference nucleotide sequences. The number of sequence reads aligned to each of the plurality of contiguous subsequences may be above a coverage threshold.

[0009] In some embodiments, determining an abundant sequence of a reference nucleotide sequence includes determining a number of sequence reads aligned to a subsequence in a plurality of subsequences of a reference nucleotide sequence in a plurality of reference nucleotide sequences. Determining an abundant sequence of a reference nucleotide sequence may include determining that an abundant sequence in an abundant sequence comprises a plurality of contiguous subsequences in a subsequence of the reference nucleotide sequence. The number of sequence reads aligned to each of the plurality of contiguous subsequences may be above a coverage threshold.

[0010] In some embodiments, one, at least one, or each of the enriched sequences comprises (i) a plurality of subsequences of a reference nucleotide sequence in a plurality of reference nucleotide sequences and (ii) interspersed subsequences of the reference nucleotide sequence that are discontinuous in the plurality of subsequences and between any two adjacent subsequences within a threshold distance of each other. The number of sequence reads aligned to each of the plurality of subsequences can be above a coverage threshold. In some embodiments, the threshold distance is about 1 nucleotide to about 50 nucleotides in length.

[0011] In some embodiments, one, at least one or each of a plurality of continuous subsequences or a plurality of subsequences has a length of one nucleotide. In some embodiments, one, at least one or each of a plurality of continuous subsequences or a plurality of subsequences has a length of at least 10 nucleotides.

[0012] In some embodiments, determining the abundant sequence of the reference nucleotide sequence includes: determining the reference nucleotide sequence in the plurality of reference nucleotide sequences each having a coverage higher than a coverage threshold. Determining the abundant sequence of the reference nucleotide sequence may include: determining that any two adjacent inferred abundant sequences of the reference nucleotide sequence in the reference nucleotide sequence are within a threshold distance on the reference nucleotide sequence. Determining the abundant sequence of the reference nucleotide sequence may include: merging two inferred abundant sequences to generate a merged inferred abundant sequence, the merged inferred abundant sequence comprising two inferred abundant sequences and interspersed subsequences of the reference nucleotide sequence between the two inferred abundant sequences. The abundant sequence may include the merged inferred abundant sequence and the inferred abundant sequence other than the merged two inferred abundant sequences. In some embodiments, the method includes: determining that any two adjacent abundant sequences of the reference nucleotide sequence in the reference nucleotide sequence are within a threshold distance on the reference nucleotide sequence; and merging the two abundant sequences to generate a merged abundant sequence, the merged abundant sequence comprising two abundant sequences and interspersed subsequences of the reference nucleotide sequence between the two abundant sequences. The merged abundant sequence may include the merged abundant sequence and the abundant sequence before merging except the merged two abundant sequences. In some embodiments, the threshold distance is from about 1 nucleotide to about 50 nucleotides in length.

[0013] In some embodiments, the highest number of coverages includes about 10 to about 500 highest number of coverages. In some embodiments, the highest number of coverages is about 1% to about 10% of the sequences of the reference nucleotide sequence with coverages above the coverage threshold. In some embodiments, the average length or median length of the sequences with coverages above the coverage threshold is about 50 to about 1000 nucleotides long. In some embodiments, at least 50% to 90% of the sequences with coverages above the coverage threshold are each at most 200 to 1000 nucleotides in length.

[0014] In some embodiments, determining the most abundant sequence of a plurality of reference nucleotide sequences having a coverage above a coverage threshold comprises: sorting the abundant sequences of a plurality of reference nucleotide sequences having a coverage above the coverage threshold in descending order of the coverage of the abundant sequences. Determining the most abundant sequence of a plurality of reference nucleotide sequences having a coverage above the coverage threshold may comprise: selecting a first abundant sequence as the most abundant sequence in descending order of the coverage of the abundant sequences. The number of the first abundant sequences in descending order of the coverage of the abundant sequences may be about 10 to about 500.

[0015] In some embodiments, no two of the most abundant sequences in the abundant sequences of the reference nucleotide sequence are within a similarity threshold of each other. In some embodiments, the method includes: determining a similarity score between each pair of the most abundant sequences. The method may include: iteratively removing each of the most abundant sequences having a similarity score higher than a similarity threshold of the remaining most abundant sequences relative to any other most abundant sequences remaining in the plurality of most abundant sequences. In some embodiments, the method includes: iteratively determining that the similarity score between the remaining pair of most abundant sequences is above a similarity threshold; and removing one of the most abundant sequence pairs from the remaining most abundant sequences. In some embodiments, the similarity threshold is about 70% to about 90%.

[0016] In some embodiments, one, at least one or each of the one or more nucleic acids comprises RNA, deoxyribonucleic acid (DNA), xenogeneic nucleic acid (XNA), or a combination thereof. XNA may comprise 1,5-anhydrohexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), fluoroarabinose nucleic acid (FANA), or a combination thereof.

[0017] In some embodiments, one or more nucleic acid probes for each of the most abundant sequences with the highest number of coverages for consuming a reference nucleotide sequence include one or more nucleic acid probes for splicing the most abundant sequences. Two adjacent probes in one or more nucleic acid probes can be separated from each other in the most abundant sequence by a splicing gap. In some embodiments, one, at least one or each sequence of one or more nucleic acid probes for each of the most abundant sequences with the highest number of coverages for consuming a reference nucleotide sequence and the most abundant sequence, its subsequence or the reverse complementary sequence of any of the foregoing have at least 80% sequence similarity. In some embodiments, the probe length can be about 25 to about 100 nucleotides long. In some embodiments, the splicing gap is about 1 to about 50 nucleotides long. In some embodiments, the average number or median number of one or more nucleic acid probes for consuming each of the most abundant sequences is about 1 to about 100. In some embodiments, the total number of probes designed to consume the most abundant sequences is less than 10,000.

[0018] In some embodiments, the sample comprises a microbial sample, a microbiome sample, a bacterial sample, a yeast sample, a plant sample, an animal sample, a patient sample, an epidemiological sample, an environmental sample, a soil sample, a water sample, a metatranscriptomics sample, or a combination thereof. In some embodiments, the sample comprises an organism of an unpredetermined species, an unknown species, or a combination thereof. In some embodiments, the sample comprises an organism of at least two species. One or more enriched RNA transcripts may include RNA transcripts from organisms of at least two species. The sample may include at least 10 ng of RNA transcripts.

[0019] In some embodiments, one or more abundant RNA transcripts, sequences thereof, or subsequences thereof are depleted from the sample using a plurality of depletion probes prior to reverse transcribing the RNA transcripts to generate complementary DNA (cDNA) and sequencing the cDNA or its product to generate a plurality of sequence reads. The one or more abundant RNA transcripts may be ribosomal RNA transcripts and / or globin mRNA transcripts. In some embodiments, the abundant RNA transcripts or any sequences thereof are not depleted from the sample.

[0020] Disclosed herein are embodiments of a system or method for designing a probe for consuming an abundant sequence of a ribonucleic acid transcript. In some embodiments, the system includes: a non-transitory memory configured to store executable instructions; and a hardware processor communicating with the non-transitory memory, the hardware processor being programmed by executable instructions to: receive a plurality of sequence reads of a ribonucleic acid (RNA) transcript or its product in a sample. The hardware processor may be programmed by executable instructions to: receive a coverage threshold, a probe length, a splicing gap, and / or a maximum number of abundant sequences for consumption. The hardware processor may be programmed by executable instructions to: compare each of a plurality of sequence reads with a reference nucleotide sequence or a subsequence thereof in a plurality of reference nucleotide sequences. The hardware processor may be programmed by executable instructions to: determine an abundant sequence of a reference nucleotide sequence or a subsequence thereof in a plurality of reference nucleotide sequences. Each of the abundant sequences may have a coverage higher than a coverage threshold. Coverage may be associated with a plurality of sequence reads aligned with an abundant sequence. The hardware processor may be programmed by executable instructions to: select the most abundant sequence with the highest number of coverages in an abundant sequence of a reference nucleotide sequence having a coverage higher than a coverage threshold. The number of most abundant sequences selected can be up to the maximum number of sequences for depletion. The hardware processor can be programmed with executable instructions to: design one or more nucleic acid probes for depleting each of the most abundant sequences with the highest number of coverages of the reference nucleotide sequence based on the sequence of the abundant sequences, the probe length, and the splicing gap. The hardware processor can be programmed with executable instructions to: output the sequence of the nucleic acid probe designed to deplete the most abundant sequences.

[0021] In some embodiments, one or more of the coverage threshold, probe length, splicing gap, and / or the maximum number of abundant sequences for depletion is a default value. In some embodiments, one or more of the coverage threshold, probe length, splicing gap, and / or the maximum number of abundant sequences for depletion is a non-default value.

[0022] In some embodiments, the hardware processor is programmed with executable instructions to: generate and / or cause display of a first user interface (UI) comprising (i) an input element for receiving a link of a plurality of sequence reads of an RNA transcript, and / or (ii) an input element for receiving a coverage threshold, a probe length, a splicing gap, and / or a maximum number of abundant sequences for consumption. The first UI may include one or more of default values ​​for the coverage threshold, the probe length, the splicing gap, and / or the maximum number of abundant sequences for consumption. (i) The plurality of sequence reads of the RNA transcript and / or (ii) the coverage threshold, the probe length, the splicing gap, and / or the maximum number of abundant sequences for consumption may be received from a user of the system via the first UI.

[0023] In some embodiments, to output the sequence of the nucleic acid probe designed to deplete the most abundant sequence, the hardware processor is programmed with executable instructions to: generate and / or cause display of a second UI comprising (a) the sequence of the designed nucleic acid probe, (b) a link to the sequence of the designed nucleic acid probe, and / or (c) an input element for receiving user input or selection to export the sequence of the designed nucleic acid probe.

[0024] In some embodiments, the reference nucleotide sequence in the plurality of reference nucleotide sequences is a reference RNA sequence of a gene. In some embodiments, the reference nucleotide sequence in the plurality of reference nucleotide sequences is a reference deoxyribonucleic acid (DNA) sequence of a gene.

[0025] In some embodiments, the coverage threshold is about 10 to about 10000. In some embodiments, the coverage of an enriched sequence in an enriched sequence is the number of sequence reads aligned to the enriched sequence. In some embodiments, the coverage of an enriched sequence in an enriched sequence is the minimum number of sequence reads aligned to each of a plurality of subsequences of the enriched sequence.

[0026] In some embodiments, one, at least one, or each of the enriched sequences comprises a plurality of contiguous subsequences of a reference nucleotide sequence in a plurality of reference nucleotide sequences. The number of sequence reads aligned to each of the plurality of contiguous subsequences may be above a coverage threshold.

[0027] In some embodiments, to determine an enriched sequence of a reference nucleotide sequence, the hardware processor is programmed with executable instructions to: determine the number of sequence reads aligned to a subsequence in a plurality of subsequences of a reference nucleotide sequence in a plurality of reference nucleotide sequences; and determine that an enriched sequence in an enriched sequence comprises a plurality of contiguous subsequences in a subsequence of the reference nucleotide sequence. The number of sequence reads aligned to each of the plurality of contiguous subsequences may be above a coverage threshold.

[0028] In some embodiments, one, at least one, or each of the enriched sequences comprises (i) a plurality of subsequences of a reference nucleotide sequence in a plurality of reference nucleotide sequences and (ii) interspersed subsequences of the reference nucleotide sequence that are discontinuous in the plurality of subsequences and between any two adjacent subsequences within a threshold distance of each other. The number of sequence reads aligned to each of the plurality of subsequences can be above a coverage threshold. In some embodiments, the threshold distance is about 1 nucleotide to about 50 nucleotides in length.

[0029] In some embodiments, one, at least one or each of a plurality of continuous subsequences or a plurality of subsequences has a length of one nucleotide. In some embodiments, one, at least one or each of a plurality of continuous subsequences or a plurality of subsequences has a length of at least 10 nucleotides.

[0030] In some embodiments, to determine the abundant sequences of a reference nucleotide sequence, the hardware processor is programmed with executable instructions to: determine the putative abundant sequences of each of the reference nucleotide sequences in a plurality of reference nucleotide sequences having a coverage higher than a coverage threshold; determine that any two adjacent putative abundant sequences of the reference nucleotide sequence in the reference nucleotide sequence are within a threshold distance on the reference nucleotide sequence; and merge the two putative abundant sequences to generate a merged putative abundant sequence, the merged putative abundant sequence comprising the two putative abundant sequences and the interspersed subsequences of the reference nucleotide sequence between the two putative abundant sequences. The abundant sequence may comprise the merged putative abundant sequence and putative abundant sequences other than the merged two putative abundant sequences. In some embodiments, the hardware processor is programmed with executable instructions to: determine that any two adjacent abundant sequences of the reference nucleotide sequence in the reference nucleotide sequence are within a threshold distance on the reference nucleotide sequence; and merge the two abundant sequences to generate a merged abundant sequence, the merged abundant sequence comprising the two abundant sequences and the interspersed subsequences of the reference nucleotide sequence between the two abundant sequences. The merged abundant sequence may comprise the merged abundant sequence and the abundant sequence before merging other than the merged two abundant sequences. In some embodiments, the threshold distance is from about 1 nucleotide to about 50 nucleotides in length.

[0031] In some embodiments, the highest number of coverages includes about 10 to about 500 highest number of coverages. In some embodiments, the highest number of coverages is about 1% to about 10% of the sequences of the reference nucleotide sequence with coverages above the coverage threshold. In some embodiments, the average length or median length of the sequences with coverages above the coverage threshold is about 50 to about 1000 nucleotides long. In some embodiments, at least 50% to 90% of the sequences with coverages above the coverage threshold are each at most 200 to 1000 nucleotides in length.

[0032] In some embodiments, to determine the most abundant sequence of a plurality of reference nucleotide sequences having a coverage greater than a coverage threshold, the hardware processor is programmed by executable instructions to: sort the abundant sequences of the plurality of reference nucleotide sequences having a coverage greater than a coverage threshold in descending order of the coverage of the abundant sequences; and select the first abundant sequence as the most abundant sequence in descending order of the coverage of the abundant sequences. The number of the first abundant sequences in descending order of the coverage of the abundant sequences can be about 10 to about 500.

[0033] In some embodiments, no two of the most abundant sequences in the abundant sequences of the reference nucleotide sequence are within a similarity threshold of each other. In some embodiments, the hardware processor is programmed by executable instructions to: determine a similarity score between each pair of the most abundant sequences; and iteratively remove each of the most abundant sequences having a similarity score higher than the similarity threshold of the remaining most abundant sequences relative to any other most abundant sequences remaining in the plurality of most abundant sequences. In some embodiments, the hardware processor is programmed by executable instructions to: iteratively determine that the similarity score between the remaining pair of most abundant sequences is above the similarity threshold; and remove one of the most abundant sequence pairs from the remaining most abundant sequences. In some embodiments, the similarity threshold is about 70% to about 90%.

[0034] In some embodiments, one, at least one, or each of the one or more nucleic acids comprises RNA, deoxyribonucleic acid (DNA), xenogeneic nucleic acid (XNA), or a combination thereof, optionally wherein the XNA comprises 1,5-anhydrohexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), fluoroarabinose nucleic acid (FANA), or a combination thereof.

[0035] In some embodiments, one or more nucleic acid probes for each of the most abundant sequences with the highest number of coverages for consuming a reference nucleotide sequence include one or more nucleic acid probes for splicing the most abundant sequences. Two adjacent probes in one or more nucleic acid probes are separated from each other in the most abundant sequence by a splicing gap. In some embodiments, one, at least one or each sequence of one or more nucleic acid probes for each of the most abundant sequences with the highest number of coverages for consuming a reference nucleotide sequence and the most abundant sequence, its subsequence or the reverse complementary sequence of any of the foregoing have at least 80% sequence similarity. In some embodiments, the probe length is about 25 to about 100 nucleotides long. In some embodiments, the splicing gap is about 1 to about 50 nucleotides long. In some embodiments, the average number or median number of one or more nucleic acid probes for consuming each of the most abundant sequences is about 1 to about 100. In some embodiments, the total number of probes designed to consume the most abundant sequences is less than 10,000.

[0036] In some embodiments, the sample comprises a microbial sample, a microbiome sample, a bacterial sample, a yeast sample, a plant sample, an animal sample, a patient sample, an epidemiological sample, an environmental sample, a soil sample, a water sample, a metatranscriptomics sample, or a combination thereof. In some embodiments, the sample comprises an organism of an unpredetermined species, an unknown species, or a combination thereof. In some embodiments, the sample comprises an organism of at least two species. One or more enriched RNA transcripts may include RNA transcripts from organisms of at least two species. The sample may include at least 10 ng of RNA transcripts.

[0037] In some embodiments, one or more abundant RNA transcripts, sequences thereof, or subsequences thereof are depleted from the sample using a plurality of depletion probes prior to reverse transcribing the RNA transcripts to generate complementary DNA (cDNA) and sequencing the cDNA or its product to generate a plurality of sequence reads. The one or more abundant RNA transcripts may be ribosomal RNA transcripts and / or globin mRNA transcripts. In some embodiments, the abundant RNA transcripts or any sequences thereof are not depleted from the sample.

[0038] Disclosed herein are embodiments of a computer-readable medium comprising executable instructions that, when executed by a hardware processor of a computing system or device, cause the hardware processor and / or the computing system or device to perform any of the methods disclosed herein. Disclosed herein are embodiments of a computer-readable medium comprising executable instructions, a non-transitory memory configured to store the executable instructions and / or the executable instructions are executed by the hardware processor of any system disclosed herein.

[0039] Disclosed herein are embodiments of compositions for depleting abundant transcripts. In some embodiments, the composition comprises: a plurality of depletion probes; and / or a plurality of supplemental depletion probes comprising nucleic acid probes designed using any of the methods or systems disclosed herein. Disclosed herein are embodiments of compositions for depleting abundant transcripts. In some embodiments, the composition comprises: a plurality of depletion probes comprising nucleic acid probes designed using any of the methods or systems disclosed herein. Disclosed herein are kits for depleting abundant transcripts. In some embodiments, the kit comprises a composition disclosed herein; and instructions for using the composition to deplete abundant transcripts.

[0040] Embodiments of methods for depleting abundant transcripts are disclosed herein. In some embodiments, the method includes: receiving a sample comprising a plurality of ribonucleic acid (RNA) transcripts. The method may include: depleting the abundant transcripts in the sample using a composition disclosed herein and one or more nucleases to generate a plurality of remaining RNA transcripts in the sample. The method may include: performing RNA sequencing on the plurality of remaining RNA transcripts in the sample to generate a plurality of sequencing reads. In some embodiments, the one or more nucleases include an RNase and / or a DNase, optionally wherein the RNase is an RNase H, and optionally wherein the DNase is a DNase 1.

[0041] Details of one or more specific implementations of the subject matter described in this specification are set forth in the accompanying drawings and the following description. Other features, aspects, and advantages will become apparent from the specification, drawings, and claims. Neither the present disclosure nor the following detailed description is intended to define or limit the scope of the subject matter of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1A-1B is a non-limiting exemplary schematic diagram showing how abundant regions of RNA transcripts in a sample can be determined.

[0043] Figure 2 is a flow chart illustrating an exemplary method for designing probes for depleting abundant sequences of RNA transcripts.

[0044] Figure 3 is a block diagram of an illustrative computing system configured to design probes for depleting abundant sequences of RNA transcripts.

[0045] Figure 4A-4B is a non-limiting exemplary graph illustrating the variable performance of a panel of 377 oligonucleotide probes in depleting rRNA and globin mRNA in different samples.

[0046] Figure 5is a non-limiting exemplary graph showing the size distribution of abundant regions in a sample after a set of 377 oligonucleotide probes were used to deplete rRNA and globin mRNA.

[0047] Figure 6 is a non-limiting exemplary heat map showing the similarity of abundant regions in samples after a set of 377 oligonucleotide probes were used to deplete rRNA and globin mRNA.

[0048] Figure 7 is a non-limiting exemplary schematic diagram showing the in silico performance of a set of 377 oligonucleotide probes and additional probes designed to deplete rRNA and globin mRNA in different samples. DETAILED DESCRIPTION

[0049] In the following detailed description, reference is made to the accompanying drawings, which form a part of the detailed description. In the accompanying drawings, similar symbols generally identify similar components unless the context otherwise dictates. The exemplary embodiments described in the detailed description, drawings, and claims are not intended to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that, as generally described herein and as shown in the accompanying drawings, various aspects of the present disclosure may be arranged, substituted, combined, separated, and designed into a variety of different configurations, all of which are expressly contemplated herein and form a part of the present disclosure.

[0050] Disclosed herein are embodiments of methods for designing probes for consuming abundant sequences of ribonucleic acid transcripts. In some embodiments, the method is controlled by a hardware processor (or a processor, such as a virtual processor) and includes: receiving a plurality of sequence reads of ribonucleic acid (RNA) transcripts or their products in a sample. The method may include: comparing each of the plurality of sequence reads with a reference nucleotide sequence or a subsequence thereof in a plurality of reference nucleotide sequences. The method may include: determining an abundant sequence of a reference nucleotide sequence or a subsequence thereof in a plurality of reference nucleotide sequences. Each of the abundant sequences may have a coverage higher than a coverage threshold. Coverage may be associated with a plurality of sequence reads aligned with the abundant sequence. The method may include: determining the most abundant sequence with the highest number of coverages in an abundant sequence of a reference nucleotide sequence having a coverage higher than a coverage threshold. The method may include: designing one or more nucleic acid probes for consuming each of the most abundant sequences with the highest number of coverages of a reference nucleotide sequence based on the sequence of the most abundant sequence, probe length, and splicing gap.

[0051] Disclosed herein is an embodiment of a system for designing probes for consuming abundant sequences of ribonucleic acid transcripts. In some embodiments, the system includes: a non-transient memory configured to store executable instructions; and a hardware processor communicating with the non-transient memory, the hardware processor being programmed by executable instructions to: receive multiple sequence reads of ribonucleic acid (RNA) transcripts or their products in a sample. The hardware processor can be programmed by executable instructions to: receive a coverage threshold, probe length, splicing gap, and / or the maximum number of abundant sequences for consumption. The hardware processor can be programmed by executable instructions to: compare each of the multiple sequence reads with a reference nucleotide sequence or a subsequence thereof in a plurality of reference nucleotide sequences. The hardware processor can be programmed by executable instructions to: determine the abundant sequences of a reference nucleotide sequence or a subsequence thereof in a plurality of reference nucleotide sequences. Each of the abundant sequences can have a coverage higher than a coverage threshold. Coverage can be related to multiple sequence reads aligned with the abundant sequence. The hardware processor can be programmed by executable instructions to: select the most abundant sequence with the highest number of coverages in the abundant sequence of the reference nucleotide sequence with a coverage higher than a coverage threshold. The number of most abundant sequences selected can be up to the maximum number of sequences for depletion. The hardware processor can be programmed with executable instructions to: design one or more nucleic acid probes for depleting each of the most abundant sequences with the highest number of coverages of the reference nucleotide sequence based on the sequence of the abundant sequences, the probe length, and the splicing gap. The hardware processor can be programmed with executable instructions to: output the sequence of the nucleic acid probe designed to deplete the most abundant sequences.

[0052] Disclosed herein are embodiments of a computer-readable medium comprising executable instructions that, when executed by a hardware processor of a computing system or device, cause the hardware processor and / or the computing system or device to perform any of the methods disclosed herein. Disclosed herein are embodiments of a computer-readable medium comprising executable instructions, a non-transitory memory configured to store the executable instructions and / or the executable instructions are executed by the hardware processor of any system disclosed herein.

[0053] Disclosed herein are embodiments of compositions for depleting abundant transcripts. In some embodiments, the composition comprises: a plurality of depletion probes; and / or a plurality of supplemental depletion probes comprising nucleic acid probes designed using any of the methods or systems disclosed herein. Disclosed herein are embodiments of compositions for depleting abundant transcripts. In some embodiments, the composition comprises: a plurality of depletion probes comprising nucleic acid probes designed using any of the methods or systems disclosed herein. Disclosed herein are kits for depleting abundant transcripts. In some embodiments, the kit comprises a composition disclosed herein; and instructions for using the composition to deplete abundant transcripts.

[0054] Embodiments of methods for depleting abundant transcripts are disclosed herein. In some embodiments, the method includes: receiving a sample comprising a plurality of ribonucleic acid (RNA) transcripts. The method may include: depleting the abundant transcripts in the sample using a composition disclosed herein and one or more nucleases to generate a plurality of remaining RNA transcripts in the sample. The method may include: performing RNA sequencing on the plurality of remaining RNA transcripts in the sample to generate a plurality of sequencing reads. In some embodiments, the one or more nucleases include an RNase and / or a DNase, optionally wherein the RNase is an RNase H, and optionally wherein the DNase is a DNase 1.

[0055] Throughout the drawings, reference numerals may be repeated to indicate corresponding relationships between referenced elements. The drawings are provided to illustrate exemplary embodiments described herein and are not intended to limit the scope of the present disclosure.

[0056] Deplete abundant sequences from a sample

[0057] One challenge of RNA sequencing for gene expression analysis is that after RNA extraction, most of the extracted material is dominated by a small number of highly abundant transcripts such as non-coding ribosomal RNA (rRNA). In total RNA samples from human blood, globin messenger RNA (mRNA) can be present at dominant levels.

[0058] Wasting the cost of sequencing these few transcripts that can dominate the instrument reading depth is generally undesirable. For example, in a human total RNA sample, rRNA can account for up to about 80%-85% of the sequencing reads. A test kit (such as RiboZero (Illumina, San Diego, CA)) may include a probe for consuming rRNA from a total RNA sample. The test kit can be used to consume rRNA and globin mRNA from a species (such as humans, yeast, plants, bacteria). Multiple test kits for different species may be needed because rRNA from different species do not have the same sequence. The farther the evolutionary distance between species, the more diverse the rRNA sequence. Therefore, the probes for hybridization and removal of abundant sequences need to be targeted to species or at least closely related species so that the test kit works well. The cost and logistics for making various test kits may be high.

[0059] Kits such as RiboZero Plus (Illumina, San Diego, CA) can include probes designed to deplete globin mRNA and rRNA from multiple species. The kit can simplify manufacturing while allowing greater flexibility in probe design. For example, the kit can be designed to deplete human, mouse and rat rRNA, human globin mRNA and rRNA from two representative bacterial species, Escherichia coli (E. coli) (Gram-negative) and Bacillus subtilis (B. subtilis) (Gram-positive). The kit can be well used to deplete globin mRNA and rRNA from the species for which the kit is designed.

[0060] However, bacteria are very diverse, and kits designed to deplete globin mRNA and rRNA of certain species may not be satisfactorily used for microbial sequencing in metatranscriptomics covering microbiome research, environmental microbiology, and epidemiology. The spectrum of bacterial species present in a sample from, for example, soil or the intestinal microbiome may not be predetermined. In addition, the bacterial species present in a sample may involve hundreds or possibly thousands of different species. Therefore, probes designed for only two representative species may not be sufficient to meet the needs of the metatranscriptomics field. In addition, there is an upper limit to the total number of probes that can be used to deplete abundant transcripts in a sample. Disclosed herein are embodiments of systems and methods for designing probes for depleting abundant sequences (e.g., abundant transcripts, such as rRNA and transposon mRNA) from a sample (such as a complex sample including a metatranscriptomics biological sample).

[0061] Probes designed to deplete abundant sequences from a sample

[0062] This article discloses a method for effective probe design, so that as many types of rich sequences as possible of the broad spectrum species present in the sample can be consumed, regardless of what species are present in the sample. The method can be used to identify and design probes for regions or sequences that are poorly consumed. The method can be used to collect, analyze and design probes for rich sequences in an unbiased manner. The method can achieve agnostic probe design for sample types (such as metatranscriptomics sample types). The method can be used to create a custom probe design tool to provide users with a simple method to remove any unwanted RNA sequences from their samples.

[0063] Bioinformatics analysis of residual rRNA can inform the feasibility of patching consumption gaps by additional or supplementary probes. In some embodiments, a probe library pool or probe set is used to process the abundant sequence reads from samples consumed by some globin mRNA and rRNA, and supplementary probes can be designed based on these abundant sequence reads. The method can be used for probe libraries to identify and design probes for poorly consumed regions or sequences. The method can be used to collect, analyze and design probes for abundant sequences in an unbiased manner. Fastq (or another format) files for each sample can be prepared using, for example, SortMeRNA (bioinfo.lifl.fr / RNA / sortmerna / ). The sample can be a metatranscriptomics sample (e.g., soil, water or microbiome sample), which can contain a wide spectrum of organisms, many of which may not yet be identified.

[0064] Globin mRNA and rRNA in the sample can be consumed by enzymatic consumption using, for example, one or more nucleases (such as RNase H and DNase 1). The probe can be an antisense deoxyribonucleic acid (DNA) oligonucleotide. Each probe can be 50 bases in length. The probe can be spliced ​​on the target, with a gap of 15 bases between the probes. The library can include, for example, 377 probes designed to target the following items: 28S, 18S, 16S, 12S, 5.8S and 5S rRNA of humans, mice and rats; five kinds of human globin mRNA; 23S and 16S rRNA of Bacillus subtilis (Gram-negative bacteria); and 23S and 16S rRNA of Escherichia coli (Gram-positive bacteria). 377 probes are referred to herein as RiboZero+ probes (Illumina, SanDiego, CA). The RNA consumption based on nuclease using 377 probes is referred to herein as RiboZero+. RiboZero+ probes and nuclease-based depletion of abundant transcripts using RiboZero+ probes have been described in PCT application PCT / US2019 / 067582, entitled "NUCLEASE-BASED RNA DEPLETION," filed on December 19, 2019, the contents of which are incorporated by reference in their entirety. In short, a DNA probe can be hybridized with an RNA transcript to form a DNA: RNA hybrid. DNA probes that do not hybridize with the RNA transcript can be removed. RNase H can be used to degrade regions of the RNA transcript in the hybrid that are hybridized with the DNA probe and RNA regions adjacent to regions of the RNA transcript in the hybrid that are hybridized with the DNA probe. DNase I can be used to degrade the remaining DNA probe in the DNA: RNA hybrid that was previously hybridized with the RNA transcript.

[0065] Sequence reads from a sample can be aligned to RNA sequences (e.g., in the publicly available Silva rRNA database) using, for example, SortMeRNA. Files containing aligned sequences can be processed using, for example, Samtools (samtools.sourceforge.net / ). Regions or sequences with high (e.g., 500-fold or more) coverage, abundance, or read counts can be identified using, for example, Bedtools2 (bedtools.readthedocs.io / en / latest / ). Figure 1A-1Bis a non-limiting exemplary schematic diagram showing how the coverage of RNA transcripts in a sample can be determined and how the abundant regions of RNA transcripts in a sample can be identified. Nearby regions or sequences can be merged (or paired). After merging, these regions or sequences can be sorted or ranked based on the coverage of the regions or sequences. Additional or supplementary probes can be designed based on or targeting the top n (e.g., 50) most abundant regions or sequences of each sample. A pairwise alignment of the top n (e.g., 50) most abundant regions or sequences can be performed using, for example, Blast (https: / / blast.ncbi.nlm.nih.gov) to remove regions that are similar to each other. A probe targeting one region may target another region with a similar sequence. If two abundant regions have an alignment or similarity score of 80% or higher, one of the two regions can be removed. Supplementary probes can be designed for the remaining regions. Each probe can be 50 bases in length. The probes can be spliced ​​on the target with a gap of 15 bases between the probes. The probes can be DNA oligonucleotides. The designed probes can be chemically synthesized. The designed probes can be added to the probe pool and / or exchanged with some of the probes in the pool without major changes to the method of depleting abundant probe sequences.

[0066] The designed probes can be used to remove abundant transcripts from total RNA samples to allow greater sensitivity and more cost-effective total RNA sequencing applications. The method can be unbiased because the abundant reads can be collected and used to design supplementary probes regardless of the species from which the abundant reads come. The absolute number of probes that can be combined and used to obtain sufficient RNA sequencing performance metrics is limited. The method can be used to design probes for efficient consumption while keeping the number of probes to a minimum.

[0067] In some embodiments, the method can be completely agnostic. The method may not require the specific species of the organism to be identified in advance. In some embodiments, the method can collect and process the rich sequences that escape consumption from the existing probes of the probe library, and allow the design of additional probes that can be used to supplement the original probe library to improve consumption performance. In some embodiments, the method allows the design of probes for wide-spectrum species, but relies on sequencing reads rather than complete rRNA sequences. In some embodiments, the method can utilize publicly available tools for comparison and data processing, and may not require complex programming. In some embodiments, the method can effectively design a limited set of probes to keep the cost and complexity of the probe library to a minimum. In some embodiments, the method can be used to design probes for consuming the rich transcripts in various sample types. These sample types can be highly complex mixtures of different species types (such as eukaryotic and prokaryotic microorganisms), such as marine sediments, soil and sludge. Other types of samples include human and mouse intestinal microbial groups.

[0068] Exemplary methods for designing probes to deplete abundant sequences from a sample

[0069] Figure 2 2 is a flow chart illustrating an exemplary method 200 for designing a probe for depleting an abundant sequence of nucleic acid (such as RNA transcripts) from a sample. The method 200 may be embodied in a set of executable program instructions stored on a computer-readable medium (such as one or more disk drives) of a computing system. For example, Figure 3 The computing system 300 shown and described in more detail below can execute a set of executable program instructions to implement the method 200. When the method 200 is initiated, the executable program instructions can be loaded into a memory such as a RAM and executed by one or more processors of the computing system 300. Figure 3 The computing system 300 shown describes the method 200, but the description is merely illustrative and is not intended to be limiting. In some embodiments, the method 200 or portions thereof may be performed serially or in parallel by multiple computing systems.

[0070] After method 200 begins at block 204, method 200 proceeds to block 208 where a computing system (e.g., Figure 3 The computing system 300 shown receives a plurality of sequence reads of nucleic acids in a sample, such as ribonucleic acid (RNA) transcripts or products thereof (eg, complementary deoxyribonucleic acid (cDNA) products from first strand synthesis).

[0071] sample. The sample may include a microbial sample, a microbiome sample, a bacterial sample, a yeast sample, a plant sample, an animal sample, a patient sample, an epidemiological sample, an environmental sample, a soil sample, a water sample, a metatranscriptomics sample, or a combination thereof. In some embodiments, the sample includes organisms of unpredetermined species, unknown or unidentified species, or a combination thereof. In some embodiments, the sample includes organisms of about, at least, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 species, or species of values ​​or ranges between any two of these values. One or more abundant RNA transcripts may include RNA transcripts from organisms of about, at least, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 species, or species of values ​​or ranges between any two of these values. A sample may contain, contain about, contain at least, or contain at most 1 ng, 2 ng, 3 ng, 4 ng, 5 ng, 6 ng, 7 ng, 8 ng, 9 ng, 10 ng, 20 ng, 30 ng, 40 ng, 50 ng, 60 ng, 70 ng, 80 ng, 90 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, 1000 ng of RNA transcripts.

[0072] User Input In some embodiments, the computing system receives a coverage threshold, probe length, splicing gap, and / or maximum number of abundant sequences for consumption from, for example, a user of the system. The computing system can retrieve the coverage threshold, probe length, splicing gap, and / or maximum number of abundant sequences for consumption from, for example, a database of the system, a memory of the system, or another system connected to the system (e.g., directly or indirectly via one or more wired or wireless networks). One or more of the coverage threshold, probe length, splicing gap, and / or maximum number of abundant sequences received and / or retrieved for consumption can be a default or non-default value.

[0073] The computing system can generate and / or cause a first user interface (UI) to be displayed. The first UI can include (i) an input element (e.g., a text box) for receiving a link of multiple sequence reads of RNA transcripts, and / or (ii) an input element (e.g., a text box and / or a drop-down list) for receiving a coverage threshold, a probe length, a splicing gap, and / or a maximum number of abundant sequences for consumption. The first UI can include one or more of the default values ​​for the coverage threshold, the probe length, the splicing gap, and / or the maximum number of abundant sequences for consumption. (i) Multiple sequence reads of RNA transcripts and / or (ii) the coverage threshold, the probe length, the splicing gap, and / or the maximum number of abundant sequences for consumption can be received from a user of the system via the first UI.

[0074] Consumption . Before the RNA transcripts are reverse transcribed to generate complementary DNA (cDNA) and the cDNA or its product is sequenced to generate multiple sequence reads, one or more abundant RNA transcripts, sequences thereof, or subsequences thereof, can be depleted from the sample using multiple depletion probes. For example, some abundant transcripts in the sample or cells in the sample may have been depleted using depletion probes. Depletion probes can be designed using the methods disclosed herein. The one or more abundant RNA transcripts can be ribosomal RNA transcripts and / or globin mRNA transcripts. In some embodiments, no abundant RNA transcripts or any sequences thereof are depleted from the sample.

[0075] Method 200 proceeds from box 208 to box 212, where the computing system compares each of the plurality of sequence reads to a reference nucleotide sequence or a subsequence thereof in a plurality of reference nucleotide sequences. The reference nucleotide sequence in the plurality of reference nucleotide sequences may be a reference RNA sequence of a gene or a subsequence thereof. The reference RNA sequence may be from the Silva rRNA database (www.arb-silva.de). The computing system may use SortMeRNA (bioinfo.lifl.fr / RNA / sortmerna / ) to compare each of the plurality of sequence reads to a reference RNA sequence or a subsequence thereof in a plurality of reference RNA sequences. The reference nucleotide sequence in the plurality of reference nucleotide sequences may be a reference deoxyribonucleic acid (DNA) sequence of a gene or a subsequence thereof.

[0076] Method 200 proceeds from box 212 to box 216, where the computing system determines an enriched sequence of a reference nucleotide sequence or a subsequence thereof in a plurality of reference nucleotide sequences. Each of the enriched sequences may have a coverage that is higher than a coverage threshold. The coverage may be associated with a plurality of sequence reads aligned with the enriched sequence. The coverage of the enriched sequence in the enriched sequence may be the number of sequence reads aligned with the enriched sequence. The coverage of the enriched sequence in the enriched sequence may be the minimum number of sequence reads aligned with each of the plurality of subsequences of the enriched sequence. The number of sequence reads aligned with each of the plurality of subsequences may be higher than the coverage threshold. In some embodiments, the coverage threshold is, is about, is at least, or is at most 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or a number or range between any two of these values.

[0077] Subsequence of a reference nucleotide sequence One, at least one or each of the enriched sequences may comprise a plurality of contiguous subsequences of a reference nucleotide sequence in a plurality of reference nucleotide sequences. The number of sequence reads aligned to each of the plurality of contiguous subsequences may be above a coverage threshold.

[0078] To determine an enriched sequence of a reference nucleotide sequence, a computing system can determine the number of sequence reads aligned to a subsequence in a plurality of subsequences of a reference nucleotide sequence in a plurality of reference nucleotide sequences (e.g., coverage). The computing system can determine that an enriched sequence in an enriched sequence comprises a plurality of contiguous subsequences in a subsequence of the reference nucleotide sequence. The number of sequence reads aligned to each of the plurality of contiguous subsequences can be above a coverage threshold.

[0079] One, at least one or each abundant sequence in the abundant sequence can comprise multiple subsequences of the reference nucleotide sequence in (i) multiple reference nucleotide sequences and (ii) interspersed subsequences between any two adjacent subsequences of the reference nucleotide sequence that are discontinuous in multiple subsequences and within a threshold distance from each other. For example, if two adjacent abundant sequences have been merged, the sequence between the two adjacent abundant sequences does not have high coverage. For example, if three adjacent abundant sequences have been merged, the resulting abundant subsequences include two interspersed subsequences between the three adjacent abundant sequences. In some embodiments, the threshold distance is, is about, is at least or is at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides long or the nucleotide length of the number or range between any two of these values.

[0080] The length of one, at least one or each of a plurality of continuous subsequences or a plurality of subsequences can be one nucleotide. For example, coverage can be calculated according to the reference sequence position. The length of one, at least one or each of a plurality of continuous subsequences or a plurality of subsequences can be, be about, be at least or be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides. For example, the coverage of a fragment of at least 10 nucleotides can be calculated.

[0081] merge . Nearby sequences can be merged. In order to determine the abundant sequences of a reference nucleotide sequence, the computing system can: determine the putative abundant sequences of each of the reference nucleotide sequences in the multiple reference nucleotide sequences that have a coverage higher than the coverage threshold. The computing system can determine that any two adjacent putative abundant sequences of the reference nucleotide sequence in the reference nucleotide sequence are within a threshold distance on the reference nucleotide sequence. The computing system can merge two putative abundant sequences to generate a merged putative abundant sequence, which contains two putative abundant sequences and interspersed subsequences of the reference nucleotide sequence between the two putative abundant sequences. The abundant sequence can contain the merged putative abundant sequence and the putative abundant sequence other than the two putative abundant sequences merged. In some embodiments, the computing system can determine that any two adjacent abundant sequences of the reference nucleotide sequence in the reference nucleotide sequence are within a threshold distance on the reference nucleotide sequence. The computing system can merge two abundant sequences to generate a merged abundant sequence, which contains two abundant sequences and interspersed subsequences of the reference nucleotide sequence between the two abundant sequences. The merged abundant sequence can contain the merged abundant sequence and the abundant sequence before the merge except for the two merged abundant sequences. In some embodiments, the threshold distance is, is about, is at least, or is at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides long, or a number or range of nucleotides long between any two of these values.

[0082] From block 216, method 200 proceeds to block 220, where the computing system determines or selects the most abundant sequence with the highest number of coverages among the abundant sequences of the reference nucleotide sequence having coverages above a coverage threshold. The number of most abundant sequences determined or selected may be up to the maximum number of sequences used for consumption.

[0083] In some embodiments, the highest number of coverages includes, includes about, includes at least, or includes at most 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 the highest number of coverages, or a number or range of the highest number of coverages between any two of these values. In some embodiments, the highest number of coverages is, is about, is at least, or is at most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or a number or range between any two of these values ​​of the reference nucleotide sequence having a coverage above the coverage threshold. In some embodiments, the average or median length of sequences having a coverage above a coverage threshold is, is about, is at least, or is at most 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 nucleotides long, or a number or range of nucleotides between any two of these values. In some embodiments, the percentage or percentage range (e.g., 50%-90%) of sequences having a coverage each above a coverage threshold is, is about, is at least, or is at most 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 nucleotides long, or a number or range of nucleotides between any two of these values. In some embodiments, the percentage or percentage range is, is about, is at least, or is at most 50%, 60%, 70%, 80%, 90%, 100%, or an amount or range between any two of these values.

[0084] Sorting . The abundant sequences can be sorted by coverage. In some embodiments, in order to determine the most abundant sequence with a coverage higher than a coverage threshold of a plurality of reference nucleotide sequences, the computing system can sort the abundant sequences with a coverage higher than the coverage threshold of a plurality of reference nucleotide sequences in descending order of the coverage of the abundant sequences. The computing system can select the first abundant sequence as the most abundant sequence in descending order of the coverage of the abundant sequences. The number of the first abundant sequence in descending order of the coverage of the abundant sequences can be, is about, is at least or is at most 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or a number or range between any two of these values.

[0085] Similar sequences. A pairwise alignment of the most abundant sequences can be performed, and the abundant sequences can be removed so that the remaining abundant sequences are dissimilar. In some embodiments, no two of the most abundant sequences in the abundant sequences of the reference nucleotide sequence are within a similarity threshold of each other. In some embodiments, the computing system can: determine a similarity score (e.g., alignment percentage) between each pair of the most abundant sequences; and iteratively remove each most abundant sequence having a similarity score higher than a similarity threshold of the remaining most abundant sequences relative to any other most abundant sequence remaining in the plurality of most abundant sequences. In some embodiments, the computing system can: iteratively determine that the similarity score between the remaining pair of most abundant sequences is above a similarity threshold; and remove one of the most abundant sequence pairs from the remaining most abundant sequences. In some embodiments, the similarity threshold is, is about, is at least, or is at most 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, or a value or range between any two of these values.

[0086] From block 220, method 200 proceeds to block 224, where the computing system designs one or more nucleic acid probes to deplete each of the most abundant sequences having the highest amount of coverage of the reference nucleotide sequence based on the sequence of the most abundant sequence, probe length, and tiling gap.

[0087] Probe. In some embodiments, one or more nucleic acid probes for each of the most abundant sequences with the highest number of coverages for consuming a reference nucleotide sequence include one or more nucleic acid probes for splicing the most abundant sequences. Two adjacent probes in one or more nucleic acid probes can be separated from each other in the most abundant sequence by a splicing gap. In some embodiments, one, at least one or each of the one or more nucleic acid probes for each of the most abundant sequences with the highest number of coverages for consuming a reference nucleotide sequence has at least 80% sequence similarity with the most abundant sequence, its subsequence or the reverse complementary sequence of any of the foregoing. In some embodiments, the sequence similarity is, is about, is at least or is at most 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% or a numerical value or range between any two of these values. In some embodiments, the probe length is, is about, is at least or is at most 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 nucleotides long, or a number or range of nucleotides between any two of these values. In some embodiments, the splicing gap is, is about, is at least or is at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 nucleotides long, or a number or range of nucleotides between any two of these values. In some embodiments, the average number or median number of one or more nucleic acid probes used to deplete each of the most abundant sequences is, is about, is at least or is at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or range between any two of these values. In some embodiments, the total number of probes designed to deplete the most abundant sequences is, is about, is at least, or is at most 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or a number or range between any two of these values.

[0088] Output. In some embodiments, the computing system outputs information related to the nucleic acid probe designed to consume the most abundant sequence. The information related to the nucleic acid probe may include the sequence of the nucleic acid probe, the coverage threshold, the probe length, the splicing gap and / or the maximum number of abundant sequences for consumption. In some embodiments, in order to output the nucleic acid probe designed to consume the most abundant sequence, the computing system may generate and / or cause a second UI to be displayed, which includes (a) the sequence of the designed nucleic acid probe, (b) a link to the sequence of the designed nucleic acid probe (e.g., a website), and / or (c) an input element (e.g., a button) for receiving user input or selection to derive the sequence of the designed nucleic acid probe.

[0089] The method 200 ends at block 228 .

[0090] Compositions and kits

[0091] Disclosed herein are embodiments of compositions for depleting abundant transcripts. In some embodiments, the composition comprises: a plurality of depletion probes; and / or a plurality of supplemental depletion probes (e.g., nucleic acid probes, such as DNA probes) designed using any of the methods or systems disclosed herein. Disclosed herein are embodiments of compositions for depleting abundant transcripts. In some embodiments, the composition comprises: a plurality of depletion probes comprising nucleic acid probes designed using any of the methods or systems disclosed herein. The depletion probes and / or the supplemental depletion probes can be single-stranded nucleic acid probes. Disclosed herein are kits for depleting abundant transcripts. In some embodiments, the kit comprises a composition disclosed herein; and instructions for using the composition to deplete abundant transcripts.

[0092] In some embodiments, one, at least one, or each of the one or more nucleic acids comprises RNA, deoxyribonucleic acid (DNA), xenogeneic nucleic acid (XNA), or a combination thereof, optionally wherein the XNA comprises 1,5-anhydrohexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), fluoroarabinose nucleic acid (FANA), or a combination thereof.

[0093] Use probes designed to deplete abundant transcripts

[0094] Embodiments of methods for consuming abundant transcripts are disclosed herein. In some embodiments, the method includes: receiving a sample containing multiple ribonucleic acid (RNA) transcripts. The method may include: using the composition disclosed herein and one or more nucleases to consume the abundant transcripts in the sample to generate multiple remaining RNA transcripts in the sample. The method may include: RNA sequencing of multiple remaining RNA transcripts in the sample to generate multiple sequencing reads. In some embodiments, the one or more nucleases include RNA enzymes and / or DNA enzymes. The RNA enzyme may be RNA enzyme H. The DNA enzyme may be DNA enzyme 1. In some embodiments, the DNA probe of the composition hybridizes with the RNA transcript to form a DNA: RNA hybrid. Excess DNA probes can be removed. RNA enzyme H can be used to degrade regions of RNA transcripts hybridized with the DNA probe in the hybrid and RNA regions adjacent to regions of RNA transcripts hybridized with the DNA probe in the hybrid. DNA enzyme I can be used to degrade the remaining DNA probes previously hybridized with the RNA transcripts in the DNA: RNA hybrid.

[0095] Execution Environment

[0096] Figure 3 Depicted is the general architecture of an exemplary computing device 300 configured to implement any of the probe design methods disclosed herein. Figure 3 The general architecture of the depicted computing device 300 includes an arrangement of computer hardware and software components. The computing device 300 may include Figure 3 310 can be connected to a computer 370, and the processing unit ...

[0097] Memory 370 may contain computer program instructions (grouped into modules or components in some embodiments) that processing unit 310 executes to implement one or more embodiments. Memory 370 typically includes RAM, ROM, and / or other persistent, secondary, or non-transitory computer-readable media. Memory 370 may store an operating system 372 that provides computer program instructions for use by processing unit 310 in general management and operation of computing device 300. Memory 370 may also include computer program instructions and other information for implementing aspects of the present disclosure.

[0098] For example, in one embodiment, memory 370 includes a probe design module 374 for designing probes, such as reference Figure 2 The method 200 for designing probes for depleting abundant sequences is described. In addition, the memory 370 may include a data repository 390 and / or one or more other data repositories storing sequencing reads used to design probes and / or designed probes or communicating with these data repositories.

[0099] Example

[0100] Certain aspects of the embodiments discussed above are disclosed in further detail in the following examples, which are not intended to limit the scope of the disclosure in any way.

[0101] Example 1

[0102] Probe design

[0103] This example demonstrates probes designed to deplete abundant sequences from a sample.

[0104] Figure 4A-4B is a non-limiting exemplary graph showing the variable performance of a set of 377 depletion probes for RiboZero and RiboZero+ in depleting rRNA and globin mRNA in different samples. A set of 377 depletion probes was used to deplete a mock colony sample ( Figure 4A ) and from several environments ( Figure 4BGlobin mRNA and rRNA in metatranscriptomic RNA samples from 100 samples of 24 samples (including marine sludge, coasts, sediments, and salt marshes). The samples were sequenced using the TruSeq (Illumina, San Diego, CA) Stranded RNA Kit. The rRNA depletion effect was better for some samples and less for others. Without being limited by theory, the different levels of depletion observed were due to regions of the bacterial rRNA that the probe could not effectively hybridize to and therefore could not effectively deplete. Figure 4A-4B RiboZero+ was shown to have higher accuracy in all samples tested, with variable nucleus depletion performance across sample types. RiboZero performed well in human skin samples and simulated communities of 20 strains, as well as environmental (bacterial) sludge samples. RiboZero+ (RNase H) had excellent performance on simulated human gut communities and environmental (bacterial) coastal and sediment samples. The RiboZero+ method has the unique ability to easily achieve performance upgrades or sample expansion.

[0105] Supplementary probes were designed for mock samples (20 strain mixtures (MSA2002)—8 replicates; skin mixtures (MSA2005)—6 replicates; and gut mixtures (MSA2006)—6 replicates) and environmental samples (coast, sediment, sludge, and salt marsh—2 replicates each) from the American Type Culture Collection. RiboZero+ probes were used to deplete abundant transcripts in the samples. The remaining rRNA sequences were sequenced using the TruSeq (Illumina, San Diego, CA) Stranded RNA Kit. Fastq (or another format) files for each sample were prepared using SortMeRNA (bioinfo.lifl.fr / RNA / sortmerna / ). Sequence reads from the samples were aligned to RNA sequences in the publicly available Silva rRNA database using SortMeRNA. Files containing aligned sequences were processed using Samtools (samtools.sourceforge.net / ). Bedtools2 (bedtools.readthedocs.io / en / latest / ) was used to identify regions or sequences with high coverage, abundance, or read counts (500-fold or more). Figure 5 is a non-limiting exemplary graph showing the size distribution of abundant regions with a coverage of at least 500 in a sample after RiboZero+ probes were used to deplete rRNA and globin mRNA. Most regions or sequences with high coverage are less than 200 nucleotides in length, such as Figure 5As shown. Merge (or pair) nearby regions or sequences. After merging, the regions or sequences are sorted or ranked based on their coverage. Additional or supplementary probes are designed to target the top 50 most abundant regions or sequences for each sample. Use Blast (https: / / blast.ncbi.nlm.nih.gov) to perform a pairwise alignment of the top 50 most abundant regions or sequences to remove regions that are similar to each other. If two abundant regions have an alignment percentage of 80% or higher, one of the two regions is removed. Figure 6 is a non-limiting exemplary heat map showing the similarity of abundant regions in samples after depletion using RiboZero+. The heat map shows blocks of similar sequences where minimal and focused probe sets can be designed. Supplementary probes are designed for the remaining regions. The probes are designed to be 50 nucleotides in length and spliced ​​on the target with a gap of 15 bases between probes. For the intestinal sample type, 50 supplementary probes are designed. For the skin sample type, 56 supplementary probes are designed. For the mixed sample type of 20 strains, 274 supplementary probes remain after pairing approximately 50 designed probes. For the intestinal sample type, skin sample type, and the mixed sample type of 20 strains, a total of 380 supplementary probes are designed. For the environmental sample type, 179 probes are designed.

[0106] After the probe sequences are generated for each sample type, they are analyzed in silico to assess how well the probes work. Figure 7 It is a non-limiting exemplary schematic diagram to determine the in-computer performance of RiboZero+ probes and supplementary probes designed to consume rRNA and globin mRNA in different samples. Blast is performed on the supplementary probe or new probe sequence relative to the Silva database. Filter the Blast results (alignment percentage is at least 80) and add a padding portion of 50 base pairs at each end. Add a padding portion at each end of the Blast hit region because it is expected that the probe will work around this region, not just where the probe binds. The "region that the new probe can consume" includes the region where each probe binds and the two padding portions at both ends of the probe. For each sample sequenced, run SortMeRNA (only keep the best hit) to obtain rRNA alignment relative to the Silva database. Use Bedtools2 to count the reads overlapping with the "region that the new probe can consume". Estimate the number of reads that were initially mapped to rRNA and then may be consumed by the new probe group. Table 1-4 shows the performance of the designed supplementary probes.

[0107] Table 1. Intestinal samples (50 supplementary probes)

[0108] sample Original rRNA content % rRNA estimates using new probes 1 15.46％ 4.13％ 2 14.58％ 3.22％ 3 14.9％ 3.7％ 4 10.87％ 3.06％ 5 11.04％ 2.96％ 6 9.15％ 1.38％

[0109] Table 2. Skin samples (56 supplementary probes)

[0110] sample Original rRNA content % rRNA estimates using new probes 1 49.68％ 6.58％ 2 52.94％ 7.31％ 3 48.66％ 6.65％ 4* 56.15％ 32.38％ 5 57.19％ 5％ 6 55.83％ 3.27％

[0111] *Sample 4 has very low yield (16k reads total compared to over 1M reads for other samples)

[0112] Table 3. Mixed samples of 20 strains (274 supplementary probes)

[0113] sample Original rRNA content % rRNA estimates using new probes 1 18.25％ 5.51％ 2 19.08％ 5.31％ 3 8.00％ 1.70％ 4 10.11％ 4.61％ 5 7.24％ 3.48％ 6 5.84％ 1.72％ 7 4.09％ 1.62％

[0114] Table 4. Environmental samples (179 supplementary probes)

[0115] sample environment Original rRNA content % rRNA estimates using new probes 1 coastal 60.23％ 40.74％ 2 coastal 61.89％ 44.03％ 3 Sediment 53.15％ 45.3％ 4 Sediment 55.30％ 48.16％ 5 Sludge 63.96％ 51.27％ 6 Sludge 63.06％ 49.94％ 7 salt marsh 52.02％ 45.81％ 8 salt marsh 42.76％ 35.36％

[0116] Together, these data demonstrate that complementary probes designed using the methods disclosed herein can have good performance in depleting abundant transcripts in different samples.

[0117] Additional considerations

[0118] In at least some of the foregoing embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment, unless such replacement is technically infeasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the above methods and structures without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter defined by the appended claims.

[0119] Those skilled in the art will appreciate that for this and other processes and methods disclosed herein, the functions performed in these processes and methods may be implemented in different orders. In addition, the steps and operations outlined are provided only as examples, and some of these steps and operations may be optional, combined into fewer steps and operations, or expanded into additional steps and operations without detracting from the essence of the disclosed embodiments.

[0120] With regard to the use of substantially any plural and / or singular terms herein, those skilled in the art may appropriately convert from plural to singular and / or from singular to plural, depending on the context and / or application. For clarity, various singular / plural arrangements may be explicitly shown herein. As used in this specification and the appended claims, the singular forms "one", "a kind of" and "the" include plural referents unless the context clearly dictates otherwise. Therefore, phrases such as "a device is configured to" are intended to include one or more of the devices. Such one or more of the devices may also be configured together to perform the statements. For example, "a processor configured to perform statements A, B, and C" may include a first processor configured to perform statement A and to work in conjunction with a second processor configured to perform statements B and C. Unless otherwise indicated, any reference to "or" herein is intended to include "and / or".

[0121] It should be understood by those skilled in the art that, in general, the terms used herein, especially in the appended claims (e.g., the bodies of the appended claims), are generally intended to be "open" terms (e.g., the term "including" should be interpreted as "including but not limited to", the term "having" should be interpreted as "having at least", the term "comprising" should be interpreted as "including but not limited to", etc.). It should also be understood by those skilled in the art that if a specific number of an introduced claim recitation is intended, such intent will be expressly stated in the claim, and in the absence of such statement, no such intent is present. For example, to aid understanding, the following appended claims may include the use of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed as implying that the introduction of a claim recitation by the indefinite article "a" or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted as meaning "at least one" or "one or more"); the same applies to the use of definite articles to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly stated, a person skilled in the art will recognize that such a statement should be interpreted as meaning at least the stated number (e.g., a direct statement of "two recitations" means at least two recitations or two or more recitations in the absence of other modifiers). Furthermore, in those cases where a convention similar to "at least one of A, B, and C, etc." is used, generally speaking, such convention is intended to be used in the sense that a person skilled in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but is not limited to systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those cases where a convention similar to "at least one of A, B, or C, etc." is used, generally speaking, such convention is intended to be used in the sense that a person skilled in the art would understand the convention (e.g., "a system having at least one of A, B, or C" would include but is not limited to systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Those skilled in the art will also understand that, in fact, any permutation of words and / or phrases presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both of the terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0122] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0123] As will be understood by those skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of their sub-ranges. Any listed range can be easily identified as fully describing and realizing that the same range is decomposed into at least equal half, one-third, one-quarter, one-fifth, one-tenth, etc. As a non-limiting example, each range discussed herein can be easily decomposed into lower third, middle third, and upper third, etc. As will be understood by those skilled in the art, all languages ​​such as "up to", "at least", "greater than", "less than", etc. include the numerical values ​​stated, and refer to the ranges that can be subsequently decomposed into sub-ranges as described above. Finally, as will be understood by those skilled in the art, ranges include each individual member. Therefore, for example, a group with 1-3 entries refers to a group with 1, 2 or 3 entries. Similarly, a group with 1-5 entries refers to a group with 1, 2, 3, 4 or 5 entries, and so on.

[0124] It should be understood that various embodiments of the present disclosure have been described herein for illustrative purposes, and various modifications may be made without departing from the scope and spirit of the present disclosure. Therefore, the various embodiments disclosed herein are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

[0125] It should be understood that not all objects or advantages may be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that certain embodiments may be configured to operate in a manner that achieves or optimizes one advantage or group of advantages as set forth herein without necessarily achieving other objects or advantages as may be set forth or suggested herein.

[0126] All of the processes described herein may be included in software code modules executed by a computing system including one or more computers or processors, and fully automated by these software code modules. The code modules may be stored in any type of non-transitory computer readable medium or other computer storage device. Some or all of the methods may be included in dedicated computer hardware.

[0127] Many other variations other than those described herein can be seen from the present disclosure. For example, according to the embodiment, some actions, events or functions of any of the algorithms described herein can be performed in different orders, can be added, merged or completely omitted (for example, not all of the actions or events are necessary for the practice of the algorithm). In addition, in certain embodiments, actions or events can be performed simultaneously and not sequentially, for example, by multithreading, interrupt processing, or multiple processors or processor cores or on other parallel architectures. In addition, different tasks or processes can be performed by different machines and / or computing systems that can run together.

[0128] The various exemplary logic blocks and modules described in conjunction with the embodiments disclosed herein may be implemented or executed by a machine designed to perform the functions described herein, such as a processing unit or processor, a digital signal processor (DSP), an application specific integrated circuit (ASIC), a field programmable gate array (FPGA) or other programmable logic device, a discrete gate or transistor logic component, a discrete hardware component, or any combination thereof. The processor may be a microprocessor, but in an alternative, the processor may be a controller, a microcontroller or a state machine, a combination thereof, or the like. The processor may include a circuit configured to process computer executable instructions. In another embodiment, the processor includes an FPGA or other programmable device that performs logic operations without processing computer executable instructions. The processor may also be implemented as a combination of computing devices, such as a combination of a DSP and a microprocessor, a plurality of microprocessors, one or more microprocessors combined with a DSP core, or any other such configuration. Although described herein primarily with respect to digital technology, the processor may also primarily include analog components. For example, some or all of the signal processing algorithms described herein may be implemented in analog circuits or mixed analog and digital circuits. By way of example, the computing environment may include any type of computer system including, but not limited to, a microprocessor-based computer system, a mainframe computer, a digital signal processor, a portable computing device, a device controller, or a computing engine within a device.

[0129] Any process description, element or frame in the flowchart described herein and / or shown in the drawings should be understood to potentially represent a module, segment or portion of code including one or more executable instructions for implementing specific logical functions or elements in the process. As will be understood by those skilled in the art, alternative specific implementations are included within the scope of the embodiments described herein, wherein elements or functions may be deleted, executed in the order shown or discussed (including substantially simultaneously or in reverse order), depending on the functions involved.

[0130] It should be emphasized that many variations and modifications may be made to the above-described embodiments, and the elements therein should be understood to be within other acceptable examples. All such modifications and variations are intended to be included within the scope of this disclosure and protected by the following claims.

Claims

1. A method for designing a probe for depleting an abundant sequence of a ribonucleic acid transcript, the method comprising: Under the control of the hardware processor: receiving a plurality of sequence reads of a ribonucleic acid (RNA) transcript or a product thereof in a sample; aligning each of the plurality of sequence reads to a reference nucleotide sequence or a subsequence thereof in a plurality of reference nucleotide sequences; determining an enriched sequence of a reference nucleotide sequence or a subsequence thereof of the plurality of reference nucleotide sequences, wherein each of the enriched sequences has a coverage above a coverage threshold related to a number of reads of the sequence aligned to the enriched sequence; determining a plurality of most abundant sequences having a highest number of coverages among the abundant sequences having coverages above the coverage threshold of the reference nucleotide sequence; as well as designing one or more nucleic acid probes for depleting each of the plurality of most abundant sequences having the highest amount of coverage of the reference nucleotide sequence based on the sequences of the plurality of most abundant sequences, probe lengths, and tile gaps; Wherein no two most abundant sequences among the abundant sequences of the reference nucleotide sequence are within a threshold of similarity to each other.

2. The method of claim 1, wherein the reference nucleotide sequence in the plurality of reference nucleotide sequences is a reference RNA sequence of a gene. 3 . The method of claim 1 , wherein a reference nucleotide sequence in the plurality of reference nucleotide sequences is a reference deoxyribonucleic acid (DNA) sequence of a gene. The method according to claim 1 , wherein the coverage threshold is 10 to 10,000.

5. The method of claim 1 , wherein the coverage of the enriched sequence in the enriched sequence is the number of sequence reads aligned with the enriched sequence, or wherein the coverage of the enriched sequence in the enriched sequence is the minimum number of sequence reads aligned with each of a plurality of subsequences of the enriched sequence.

6. A method according to claim 1, wherein at least one of the enriched sequences comprises multiple contiguous subsequences of a reference nucleotide sequence in the multiple reference nucleotide sequences, and wherein the number of sequence reads aligned to each of the multiple contiguous subsequences is higher than the coverage threshold.

7. The method of claim 1, wherein determining the abundant sequences of the reference nucleotide sequence comprises: determining a number of the sequence reads aligned to a subsequence in a plurality of subsequences of a reference nucleotide sequence in the plurality of reference nucleotide sequences; as well as It is determined that an enriched sequence in the enriched sequences comprises a plurality of contiguous subsequences in the subsequence of the reference nucleotide sequence, wherein the number of the sequence reads aligned to each of the plurality of contiguous subsequences is above the coverage threshold.

8. The method of claim 1 , wherein at least one of the enriched sequences comprises (i) a plurality of subsequences of a reference nucleotide sequence among the plurality of reference nucleotide sequences and (ii) interspersed subsequences of the reference nucleotide sequence between any two adjacent subsequences that are discontinuous among the plurality of subsequences and are within a threshold distance from each other, and wherein the number of sequence reads aligned to each of the plurality of subsequences is higher than the coverage threshold.

9. The method according to any one of claims 5 to 8, wherein the length of the plurality of consecutive subsequences or at least one of the plurality of subsequences is one nucleotide.

10. The method according to any one of claims 5 to 8, wherein the plurality of contiguous subsequences or at least one of the plurality of subsequences has a length of at least 10 nucleotides.

11. The method of claim 1, wherein determining the abundant sequences of the reference nucleotide sequence comprises: determining a putatively abundant sequence for each of the reference nucleotide sequences in the plurality of reference nucleotide sequences having a coverage above the coverage threshold; determining that any two adjacent putatively abundant sequences of a reference nucleotide sequence in the reference nucleotide sequence are within a threshold distance on the reference nucleotide sequence; as well as The two putative abundant sequences are merged to generate a merged putative abundant sequence, the merged putative abundant sequence comprising the two putative abundant sequences and an interspersed subsequence of the reference nucleotide sequence between the two putative abundant sequences, wherein the abundant sequence comprises the merged putative abundant sequence and the putative abundant sequence excluding the merged two putative abundant sequences.

12. The method according to claim 1, comprising: determining that any two adjacent abundant sequences of a reference nucleotide sequence in the reference nucleotide sequence are within a threshold distance on the reference nucleotide sequence; as well as The two abundant sequences are merged to generate a merged abundant sequence, the merged abundant sequence comprising the two abundant sequences and an interspersed subsequence of the reference nucleotide sequence between the two abundant sequences, wherein the abundant sequence after the merging comprises the merged abundant sequence and the abundant sequence before the merging excluding the merged two abundant sequences.

13. The method of claim 8, wherein the threshold distance is 1 nucleotide to 50 nucleotides long.

14. The method of claim 1, wherein the highest number of coverages comprises 10 to 500 highest number of coverages.

15. The method of claim 1, wherein the highest number of coverages is 1% to 10% of the sequences of the reference nucleotide sequence having coverage above the coverage threshold.

16. The method of claim 1, wherein the average or median length of the sequences having a coverage above the coverage threshold is 50 to 1000 nucleotides long.

17. The method of claim 1, wherein at least 50% to 90% of the sequences having a coverage above the coverage threshold are each at most 200 to 1000 nucleotides in length.

18. The method of claim 1, wherein determining the plurality of most abundant sequences of the plurality of reference nucleotide sequences having coverage above the coverage threshold comprises: sorting the enriched sequences of the plurality of reference nucleotide sequences having coverages above the coverage threshold in descending order of the coverages of the enriched sequences; as well as A first abundant sequence is selected as the plurality of most abundant sequences in the descending order of the coverage of the abundant sequences.

19. The method of claim 18, wherein the number of the first abundant sequences in the descending order of the coverage of the abundant sequences is 10 to 500.

20. The method according to claim 1, comprising: determining a similarity score between each pair of the plurality of most abundant sequences; as well as Each most abundant sequence having a similarity score greater than a similarity threshold for the remaining plurality of most abundant sequences is iteratively removed relative to any other plurality of most abundant sequences remaining in the plurality of most abundant sequences.

21. The method according to claim 1, comprising: Iteratively determining that a similarity score between a remaining pair of the plurality of most abundant sequences is above a similarity threshold; as well as One of the pairs of the plurality of most abundant sequences is removed from the remaining plurality of most abundant sequences.

22. The method of claim 1, wherein the similarity threshold is 70% to 90%.

23. The method of claim 1, wherein at least one of the one or more nucleic acid probes comprises RNA, deoxyribonucleic acid (DNA), xenogeneic nucleic acid (XNA), or a combination thereof.

24. The method of claim 23, wherein the XNA comprises 1,5-anhydrohexitol nucleic acid, cyclohexene nucleic acid (CeNA), threose nucleic acid, glycol nucleic acid, locked nucleic acid (LNA), peptide nucleic acid (PNA), fluoroarabinose nucleic acid, or a combination thereof.

25. A method according to claim 1, wherein the one or more nucleic acid probes for each of the multiple most abundant sequences having the highest number of coverages for depleting the reference nucleotide sequence include one or more nucleic acid probes for splicing the multiple most abundant sequences, and wherein two adjacent probes of the one or more nucleic acid probes are separated from each other in the multiple most abundant sequences by the splicing gap.

26. A method according to claim 1, wherein the sequence of at least one of the one or more nucleic acid probes used to deplete each of the multiple most abundant sequences with the highest amount of coverage of the reference nucleotide sequence has at least 80% sequence similarity with the multiple most abundant sequences, their subsequences, or the reverse complement sequences of the multiple most abundant sequences or their subsequences.

27. The method of claim 1, wherein the probe length is 25 to 100 nucleotides long and / or the splice gap is 1 to 50 nucleotides long.

28. The method of claim 1, wherein the average or median number of the one or more nucleic acid probes used to deplete each of the plurality of most abundant sequences is between 1 and 100.

29. The method of claim 1, wherein the total number of probes designed to deplete the plurality of most abundant sequences is less than 10,000.

30. The method of claim 1, wherein the sample comprises a microbial sample, a plant sample, an animal sample, a patient sample, an environmental sample, a metatranscriptomics sample, or a combination thereof.

31. The method of claim 1, wherein the sample comprises a microbiome sample.

32. The method of claim 1, wherein the sample comprises a bacterial sample or a yeast sample.

33. The method of claim 1, wherein the sample comprises an epidemiological sample.

34. The method of claim 1, wherein the sample comprises a soil sample or a water sample.

35. The method of claim 1, wherein the sample comprises organisms of an undetermined species.

36. The method of claim 1, wherein the sample comprises an organism of an unknown species.

37. The method of claim 1, wherein the sample comprises organisms of at least two species and / or wherein the one or more abundant RNA transcripts comprise RNA transcripts from organisms of at least two species.

38. The method of claim 1, wherein the sample comprises at least 10 ng of RNA transcripts.

39. The method of claim 1, wherein the RNA transcripts or subsequences thereof are depleted from the sample using a plurality of depleting probes prior to reverse transcribing one or more abundant RNA transcripts to generate complementary DNA (cDNA) and sequencing the cDNA or its products to generate the plurality of sequence reads.

40. The method of claim 39, wherein the one or more abundant RNA transcripts are ribosomal RNA transcripts and / or globin mRNA transcripts.

41. The method of claim 1, wherein abundant RNA transcripts or any sequences thereof are not depleted from the sample.

42. A computer system for designing probes for depleting abundant sequences of ribonucleic acid transcripts, the computer system comprising: a non-transitory memory configured to store executable instructions; as well as a hardware processor in communication with the non-transitory memory, the hardware processor being programmed by the executable instructions to: receiving a plurality of sequence reads of a ribonucleic acid (RNA) transcript or a product thereof in a sample; receiving coverage thresholds, probe lengths, splicing gaps, and / or maximum numbers of abundant sequences for depletion; aligning each of the plurality of sequence reads to a reference nucleotide sequence or a subsequence thereof in a plurality of reference nucleotide sequences; determining an enriched sequence of a reference nucleotide sequence or a subsequence thereof of the plurality of reference nucleotide sequences, wherein each of the enriched sequences has a coverage above the coverage threshold related to the number of reads of the sequence aligned to the enriched sequence; selecting a plurality of most abundant sequences having the highest number of coverages among the abundant sequences of the reference nucleotide sequence having coverages above the coverage threshold, wherein the number of the plurality of most abundant sequences selected is at most the maximum number of sequences for consumption; designing one or more nucleic acid probes to deplete each of the plurality of most abundant sequences having the highest amount of coverage of the reference nucleotide sequence based on the sequence of the abundant sequences, the probe length, and the splicing gap; as well as outputting the sequences of the nucleic acid probes designed to deplete the plurality of most abundant sequences; Wherein no two most abundant sequences among the abundant sequences of the reference nucleotide sequence are within a threshold of similarity to each other.

43. The computer system of claim 42, wherein one or more of the coverage threshold, the probe length, the splicing gap, and / or the maximum number of the abundant sequences for depletion is a default value, or wherein one or more of the coverage threshold, the probe length, the splicing gap, and / or the maximum number of the abundant sequences for depletion is a non-default value.

44. A computer system according to claim 42, wherein the hardware processor is programmed by the executable instructions to: generate and / or cause display of a first user interface (UI), the first user interface comprising (i) an input element for receiving a link of the multiple sequence reads of RNA transcripts, and / or (ii) an input element for receiving the coverage threshold, the probe length, the splicing gap and / or the maximum number of the abundant sequences for consumption, and wherein (i) the multiple sequence reads of RNA transcripts and / or (ii) the coverage threshold, the probe length, the splicing gap and / or the maximum number of the abundant sequences for consumption are received from a user of the system via the first UI.

45. A computer system according to claim 42, wherein, in order to output the sequence of the nucleic acid probe designed to consume the multiple most abundant sequences, the hardware processor is programmed by the executable instructions to: generate and / or cause a second UI to be displayed, the second UI comprising (a) the sequence of the designed nucleic acid probe, (b) a link to the sequence of the designed nucleic acid probe, and / or (c) an input element for receiving user input or selection to export the sequence of the designed nucleic acid probe.

46. ​​The computer system of claim 42, wherein a reference nucleotide sequence in the plurality of reference nucleotide sequences is a reference RNA sequence of a gene.

47. The computer system of claim 42, wherein a reference nucleotide sequence in the plurality of reference nucleotide sequences is a reference deoxyribonucleic acid (DNA) sequence of a gene.

48. The computer system of claim 42, wherein the coverage threshold is between 10 and 10,000.

49. A computer system according to claim 42, wherein the coverage of the enriched sequence in the enriched sequence is the number of sequence reads aligned with the enriched sequence, or wherein the coverage of the enriched sequence in the enriched sequence is the minimum number of sequence reads aligned with each of multiple subsequences of the enriched sequence.

50. A computer system according to claim 42, wherein at least one of the enriched sequences comprises multiple contiguous subsequences of a reference nucleotide sequence in the multiple reference nucleotide sequences, and wherein the number of sequence reads aligned to each of the multiple contiguous subsequences is higher than the coverage threshold.

51. The computer system of claim 42, wherein to determine the abundant sequences of the reference nucleotide sequence, the hardware processor is programmed by the executable instructions to: determining a number of the sequence reads aligned to a subsequence in a plurality of subsequences of a reference nucleotide sequence in the plurality of reference nucleotide sequences; and It is determined that an enriched sequence in the enriched sequences comprises a plurality of contiguous subsequences in the subsequence of the reference nucleotide sequence, wherein the number of the sequence reads aligned to each of the plurality of contiguous subsequences is above the coverage threshold.

52. A computer system according to claim 42, wherein at least one of the enriched sequences comprises (i) multiple subsequences of a reference nucleotide sequence in the multiple reference nucleotide sequences and (ii) interspersed subsequences of the reference nucleotide sequence between any two adjacent subsequences that are not continuous in the multiple subsequences and are within a threshold distance from each other, and wherein the number of sequence reads aligned to each of the multiple subsequences is higher than the coverage threshold.

53. The computer system of any one of claims 49-52, wherein the plurality of contiguous subsequences or at least one of the plurality of subsequences is one nucleotide in length.

54. The computer system of any one of claims 49-52, wherein the plurality of contiguous subsequences or at least one of the plurality of subsequences is at least 10 nucleotides in length.

55. The computer system of claim 42, wherein to determine the abundant sequences of the reference nucleotide sequence, the hardware processor is programmed by the executable instructions to: determining a putatively abundant sequence for each of the reference nucleotide sequences in the plurality of reference nucleotide sequences having a coverage above the coverage threshold; determining that any two adjacent putatively abundant sequences of a reference nucleotide sequence in the reference nucleotide sequence are within a threshold distance on the reference nucleotide sequence; as well as The two putative abundant sequences are merged to generate a merged putative abundant sequence, the merged putative abundant sequence comprising the two putative abundant sequences and an interspersed subsequence of the reference nucleotide sequence between the two putative abundant sequences, wherein the abundant sequence comprises the merged putative abundant sequence and the putative abundant sequence excluding the merged two putative abundant sequences.

56. The computer system of claim 42, wherein the hardware processor is programmed by the executable instructions to: determining that any two adjacent abundant sequences of a reference nucleotide sequence in the reference nucleotide sequence are within a threshold distance on the reference nucleotide sequence; and The two abundant sequences are merged to generate a merged abundant sequence, the merged abundant sequence comprising the two abundant sequences and an interspersed subsequence of the reference nucleotide sequence between the two abundant sequences, wherein the abundant sequence after the merging comprises the merged abundant sequence and the abundant sequence before the merging excluding the merged two abundant sequences.

57. The computer system of claim 52, wherein the threshold distance is 1 nucleotide to 50 nucleotides long.

58. The computer system of claim 42, wherein the highest number of coverages comprises 10 to 500 highest number of coverages.

59. The computer system of claim 42, wherein the highest number of coverages is 1% to 10% of the sequences of the reference nucleotide sequence having coverage above the coverage threshold.

60. The computer system of claim 42, wherein the average or median length of the sequences having a coverage above the coverage threshold is 50 to 1000 nucleotides long.

61. The computer system of claim 42, wherein at least 50% to 90% of the sequences having coverage above the coverage threshold are each at most 200 to 1000 nucleotides in length.

62. The computer system of claim 42, wherein to select the plurality of most abundant sequences of the plurality of reference nucleotide sequences having coverage above the coverage threshold, the hardware processor is programmed by the executable instructions to: sorting the enriched sequences of the plurality of reference nucleotide sequences having coverage above the coverage threshold in descending order of the coverage of the enriched sequences; and A first abundant sequence is selected as the plurality of most abundant sequences in the descending order of the coverage of the abundant sequences.

63. The computer system of claim 62, wherein the number of the first abundant sequences in the descending order of the coverage of the abundant sequences is between 10 and 500.

64. The computer system of claim 42, the hardware processor being programmed by the executable instructions to: determining a similarity score between each pair of the plurality of most abundant sequences; and Each most abundant sequence having a similarity score greater than a similarity threshold for the remaining plurality of most abundant sequences is iteratively removed relative to any other plurality of most abundant sequences remaining in the plurality of most abundant sequences.

65. The computer system of claim 42, wherein the hardware processor is programmed by the executable instructions to: iteratively determining that a similarity score between a remaining pair of the plurality of most abundant sequences is above a similarity threshold; and One of the plurality of most abundant sequence pairs is removed from the remaining plurality of most abundant sequences.

66. A computer system according to claim 64 or 65, wherein the similarity threshold is 70% to 90%.

67. The computer system of claim 42, wherein at least one of the one or more nucleic acid probes comprises RNA, deoxyribonucleic acid (DNA), xenogeneic nucleic acid (XNA), or a combination thereof.

68. The computer system of claim 67, wherein the XNA comprises 1,5-anhydrohexitol nucleic acid, cyclohexene nucleic acid (CeNA), threose nucleic acid, glycol nucleic acid, locked nucleic acid (LNA), peptide nucleic acid (PNA), fluoroarabinose nucleic acid, or a combination thereof.

69. A computer system according to claim 42, wherein the one or more nucleic acid probes for each of the multiple most abundant sequences with the highest number of coverages for depleting the reference nucleotide sequence include one or more nucleic acid probes for splicing the multiple most abundant sequences, and wherein two adjacent probes of the one or more nucleic acid probes are separated from each other in the multiple most abundant sequences by the splicing gap.

70. A computer system according to claim 42, wherein the sequence of at least one of the one or more nucleic acid probes used to deplete each of the multiple most abundant sequences with the highest amount of coverage of the reference nucleotide sequence has at least 80% sequence similarity with the multiple most abundant sequences, their subsequences, or the reverse complement sequences of the multiple most abundant sequences or their subsequences.

71. The computer system of claim 42, wherein the probe length is 25 to 100 nucleotides long and / or the splice gap is 1 to 50 nucleotides long.

72. The computer system of claim 42, wherein the average or median number of the one or more nucleic acid probes used to deplete each of the plurality of most abundant sequences is between 1 and 100.

73. The computer system of claim 42, wherein the total number of said probes designed to deplete said plurality of most abundant sequences is less than 10,000.

74. The computer system of claim 42, wherein the sample comprises a microbial sample, a plant sample, an animal sample, a patient sample, an environmental sample, a metatranscriptomics sample, or a combination thereof.

75. The computer system of claim 42, wherein the sample comprises a microbiome sample.

76. The computer system of claim 42, wherein the sample comprises a bacterial sample or a yeast sample.

77. The computer system of claim 42, wherein the sample comprises an epidemiological sample.

78. The computer system of claim 42, wherein the sample comprises a soil sample or a water sample.

79. The computer system of claim 42, wherein the sample comprises an organism of an undetermined species.

80. The computer system of claim 42, wherein the sample comprises an organism of an unknown species.

81. The computer system of claim 42, wherein the sample comprises organisms of at least two species and / or wherein the one or more abundant RNA transcripts comprise RNA transcripts from organisms of at least two species.

82. The computer system of claim 42, wherein the sample comprises at least 10 ng of RNA transcripts.

83. The computer system of claim 42, wherein the RNA transcripts or subsequences thereof are depleted from the sample using a plurality of depleting probes prior to reverse transcribing the one or more abundant RNA transcripts to generate complementary DNA (cDNA) and sequencing the cDNA or products thereof to generate the plurality of sequence reads.

84. The computer system of claim 83, wherein the one or more abundant RNA transcripts are ribosomal RNA transcripts and / or globin mRNA transcripts.

85. The computer system of claim 42, wherein abundant RNA transcripts or any sequences thereof are not depleted from the sample.

Images