SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 18, 2021, is named 51024-705_302_SL.txt and is 8,795 bytes in size.

BACKGROUND

The goal to elucidate the entire human genome has created interest in technologies for rapid nucleic acid (e.g., DNA) sequencing, both for small and large scale applications. As knowledge of the genetic basis for human diseases increases, high-throughput DNA sequencing has been leveraged for myriad clinical applications. Despite the prevalence of nucleic acid sequencing methods and systems in a wide range of molecular biology and diagnostics applications, such methods and systems may encounter challenges in accurate base calling, such as when sequencing signals include regions of repeating nucleotide bases called homopolymers. In particular, sequencing methods that perform base calling based on quantified characteristic signals indicating nucleotide incorporation can have sequencing errors (e.g., in quantifying homopolymer lengths), stemming from random and unpredictable systematic variations in signal levels and context dependent signals that may be different for every sequence. Such signal variations and context dependency signals may cause issues with sequence (e.g., homopolymer) calling.

SUMMARY

Recognized herein is a need for improved base calling of sequences, such as sequences containing homopolymers. Methods and systems provided herein can significantly reduce or eliminate errors in quantifying homopolymer lengths and errors associated with context dependence. Such methods and systems may achieve accurate and efficient base calling of sequences (such as sequences containing homopolymers), quantification of homopolymer lengths, and quantification of context dependency in sequence signals.

In an aspect, disclosed herein is a method for sequencing a nucleic acid molecule, comprising: (a) sequencing said nucleic acid molecule to generate a plurality of sequence signals; and (b) determining base calls of said nucleic acid molecule based at least in part on (i) said plurality of sequence signals and (ii) quantified context dependency for at least a portion of said plurality of sequence signals. In some embodiments, said sequencing generates one or more imputed sequences, and said base calls of said nucleic acid molecule are determined based at least in part on (i) said one or more imputed sequences, and (ii) quantified context dependency for at least a portion of said one or more imputed sequences. In some embodiments, (b) is performed in real time with said sequencing of (a). In some embodiments, said one or more imputed sequences comprise one or more homopolymers of length N. In some embodiments, N is at least 1 base, at least 2 bases, or at least 3 bases. In some embodiments, prior to (b), said plurality of sequence signals are pre-processed to remove systematic errors. In some embodiments, the method further comprises, prior to (b), pre-processing said plurality of sequence signals to remove systematic error(s). In some embodiments, the method further comprises determining lengths of said homopolymers based at least on clustering of said homopolymers or associated signals.

In some embodiments, said plurality of sequence signals is generated by sequencing nucleic acids of a subject. In some embodiments, said plurality of sequence signals is generated by massively parallel array sequencing. In some embodiments, said plurality of sequence signals is generated by flow sequencing.

In another aspect, disclosed herein is a method for quantifying context dependency of a plurality of sequence signals, comprising: (a) sequencing deoxyribonucleic acid (DNA) molecule(s) to provide a plurality of sequence signals and one or more imputed sequences, wherein said DNA molecule(s) comprise a known sequence; and (b) for a given locus of a plurality of loci, determining one or more expected signals for said given locus based at least on (i) said plurality of sequence signals and/or said one or more imputed sequences, and (ii) said known sequence. In some embodiments, the method further comprises: (c) sequencing a second set of DNA molecules comprising unknown sequences, thereby generating a second plurality of sequence signals; and (d) determining base calls of said second set of DNA molecules based at least in part on (i) said second plurality of sequence signals, and (ii) at least a portion of said one or more expected signals. In some embodiments, sequencing said second set of DNA molecules generates one or more imputed sequences, and said base calls of said second set of DNA molecules are determined based at least in part on said one or more imputed sequences.

In some embodiments, said DNA molecules comprise synthetic templates. In some embodiments, said known sequence comprises one or more homopolymers of length N. In some embodiments, N is at least 1 base, at least 2 bases, or at least 3 bases. In some embodiments, said one or more imputed sequences comprise one or more homopolymers of length N. In some embodiments, N is at least 1 base, at least 2 bases, or at least 3 bases. In some embodiments, quantifying said context dependency comprises establishing a context specific mapping between signal amplitudes and known sequences for each of a plurality of loci. In some embodiments, said context dependency is quantified based on a distribution of said sequence signals or of homopolymer lengths of said imputed sequences. In some embodiments, prior to (b), said plurality of sequence signals are pre-processed to remove systematic errors. In some embodiments, the method further comprises, prior to (b), pre-processing said plurality of sequence signals to remove systematic error(s). In some embodiments, the method further comprises determining lengths of said homopolymers based at least on clustering of said homopolymers or associated signals.

In some embodiments, said plurality of sequence signals is generated by sequencing nucleic acids of a subject. In some embodiments, said plurality of sequence signals is generated by massively parallel array sequencing. In some embodiments, said plurality of sequence signals is generated by flow sequencing. In some embodiments, said base calls are determined based at least on a context dependency of said second plurality of sequence signals. In some embodiments, said base calls are determined based at least on a context dependency of said second plurality of sequence signals and said one or more imputed sequences. In some embodiments, (d) is performed in real time with said sequencing of (c).

In another aspect, disclosed herein is a method for processing a plurality of sequence signals, comprising: (a) sequencing a nucleic acid sample to provide a plurality of sequence signals and imputed sequences; (b) truncating each identified imputed homopolymer sequence of at least N bases of said plurality of imputed sequences to a homopolymer sequence of bases of length N, to yield one or more HpN truncated sequences; (c) aligning said one or more HpN truncated sequences to a truncated reference(s), which truncated reference(s) has been HpN truncated and thereby comprises one or more homopolymer sequences truncated to said length N; and (d) generating a consensus sequence from said one or more HpN truncated sequences aligned to said HpN truncated reference(s), which consensus sequence comprises a homopolymer sequence of said length N, based at least on said one or more HpN truncated sequences aligned to said HpN truncated reference(s) and/or sequence signals associated with said one or more HpN truncated sequences aligned to said HpN truncated reference(s).

In some embodiments, N is 5 bases. In some embodiments, N is 6 bases. In some embodiments, N is 7 bases. In some embodiments, N is 8 bases. In some embodiments, a length estimation error of said homopolymer sequence is calculated based at least on a distribution of signals or imputed homopolymer lengths of said one or more HpN truncated sequences aligned to said HpN truncated reference(s). In some embodiments, prior to (b), said plurality of sequence signals are pre-processed to remove systematic error(s). In some embodiments, the method further comprises, prior to (b), pre-processing said plurality of sequence signals to remove systematic error(s). In some embodiments, the method further comprises determining lengths of said homopolymer sequences based at least on clustering of said homopolymer sequences or associated signals.

In some embodiments, said plurality of sequence signals is generated by sequencing nucleic acids of a subject, and said HpN truncated reference(s) comprises an HpN truncated reference genome of a species of said subject. In some embodiments, a number of lengths computed or classified in (d) is restricted based at least on the ploidy of said species of said subject. In some embodiments, said plurality of sequence signals and imputed sequences is generated by massively parallel array sequencing. In some embodiments, said plurality of sequence signals and imputed sequences is generated by flow sequencing.

In another aspect, disclosed herein is a method for quantifying context dependency of a plurality of sequence signals and imputed sequences, the method comprising: (a) sequencing deoxyribonucleic acid (DNA) molecule(s) to provide a plurality of sequence signals and imputed sequences, wherein said DNA molecule(s) comprise a known sequence; (b) truncating each identified imputed homopolymer sequence of at least N bases of said plurality of imputed sequences to a homopolymer sequence of bases of length N, to yield one or more HpN truncated sequences; (c) aligning said one or more HpN truncated sequences to a truncated reference(s), which truncated reference(s) has been HpN truncated and thereby comprises one or more homopolymer sequences HpN truncated to said length N; and (d) quantifying said context dependency of associated sequence signals based at least on (i) said one or more HpN truncated sequences aligned to said HpN truncated reference(s) and/or sequence signals associated with said one or more HpN truncated sequences aligned to said HpN truncated reference(s), and (ii) said known sequence. In some embodiments, the method further comprises: (e) sequencing a second set of DNA molecules comprising unknown sequences, thereby generating a second plurality of sequence signals and imputed sequences; (f) truncating each identified imputed second homopolymer sequence of at least N bases of said second plurality of imputed sequences to a homopolymer sequence of bases of length N, to yield one or more second HpN truncated sequences; (g) aligning said one or more second HpN truncated sequences to said HpN truncated reference(s); and (h) determining homopolymer lengths of said second plurality of DNA molecules based at least on (i) said one or more HpN truncated sequences aligned to said HpN truncated reference(s) and/or sequence signals associated with said one or more HpN truncated sequences aligned to said HpN truncated reference(s) and (ii) said quantified context dependency.

In some embodiments, said quantified context dependency is classified for a given context. In some embodiments, said given context is an n-base context, wherein ‘n’ is a number greater than or equal to 5. In some embodiments, said DNA molecule(s) is derived from a ribonucleic acid (RNA) molecule(s). In some embodiments, said plurality of sequence signals and imputed sequences is generated by massively parallel array sequencing. In some embodiments, said plurality of sequence signals and imputed sequences is generated by flow sequencing. In some embodiments, quantifying said context dependency comprises establishing a context specific mapping between signal amplitudes and homopolymer length for each of a plurality of loci.

In another aspect, disclosed herein is a method for quantifying context dependency of a plurality of sequence signals and imputed sequences, the method comprising: (a) sequencing deoxyribonucleic acid (DNA) molecule(s) to provide a plurality of sequence signals and imputed sequences, wherein said DNA molecule(s) comprise a known sequence; (b) truncating each identified imputed homopolymer sequence of at least N bases of said plurality of imputed sequences to a homopolymer sequence of bases of length N, to yield one or more HpN truncated sequences; (c) aligning said one or more HpN truncated sequences to a truncated reference(s), which truncated reference(s) has been HpN truncated and thereby comprises one or more homopolymer sequences truncated to said length N; and (d) for each of a plurality of loci in said HpN truncated reference(s), determining an expected signal for said locus based at least on (i) said one or more HpN truncated sequences aligned to said HpN truncated reference(s) and/or sequence signals associated with said one or more HpN truncated sequences aligned to said HpN truncated reference(s), and (ii) said known sequence. In some embodiments, the method further comprises: (e) sequencing a second set of DNA molecules comprising unknown sequences, thereby generating a second plurality of sequence signals and imputed sequences; (f) truncating each identified imputed second homopolymer sequence of at least N bases of said second plurality of imputed sequences to a homopolymer sequence of bases of length N, to yield one or more second HpN truncated sequences; (g) aligning said one or more second HpN truncated sequences to said HpN truncated reference(s); and (h) determining homopolymer lengths of said second set of DNA molecules based at least on (i) said one or more HpN truncated sequences aligned to said HpN truncated reference(s) and/or sequence signals associated with said one or more HpN truncated sequences aligned to said HpN truncated reference(s) and (ii) said known sequence.

In some embodiments, said DNA molecule(s) is derived from a ribonucleic acid molecule(s). In some embodiments, said plurality of sequence signals and imputed sequences is generated by massively parallel array sequencing. In some embodiments, said plurality of sequence signals and imputed sequences is generated by flow sequencing. In some embodiments, quantifying said context dependency comprises establishing a context specific mapping between signal amplitudes and homopolymer length for each of a plurality of loci.

In another aspect, disclosed herein is a method for processing a plurality of sequence signals, comprising: (a) sequencing a nucleic acid sample to provide a plurality of sequence signals and imputed sequences; (b) processing said plurality of sequence signals and imputed sequences to determine a set of one or more sequences comprising homopolymer sequences; (c) processing said plurality of sequence signals and imputed sequences to identify a presence and an estimated length of at least a portion of said homopolymer sequences; and (d) refining said estimated lengths using secondary assay data. In some embodiments, said plurality of sequence signals and imputed sequences is generated by massively parallel array sequencing. In some embodiments, said plurality of sequence signals and imputed sequences is generated by flow sequencing.

In another aspect, disclosed herein is a method for processing a plurality of sequence signals, comprising: (a) sequencing a nucleic acid sample to provide said plurality of sequence signals; (b) aligning said plurality of sequence signals to a reference signal; (c) identifying a reference locus comprising a homopolymer sequence based at least on said aligned sequence signals; and (d) generating a consensus sequence from said plurality of sequence signals aligned to said reference signal, which consensus sequence comprises a homopolymer sequence of N bases, based at least on said identified reference locus, a length of said homopolymer sequence of said reference locus, and said reference signal.

In some embodiments, a length estimation error of said homopolymer sequence is calculated based at least on a distribution of signals or imputed homopolymer lengths of said plurality of sequence signals aligned to said reference signal. In some embodiments, prior to (b), said plurality of sequence signals is pre-processed to remove systematic error(s). In some embodiments, the method further comprises, prior to (b), pre-processing said plurality of sequence signals to remove systematic error(s). In some embodiments, said plurality of sequence signals is generated by sequencing nucleic acids of a subject, and said reference signal comprises a reference genome of a species of said subject. In some embodiments, a number of lengths computed or classified is restricted based at least on the ploidy of said species of said subject. In some embodiments, said plurality of sequence signals is generated by massively parallel array sequencing. In some embodiments, said plurality of sequence signals is generated by flow sequencing.

In another aspect, disclosed herein is a method for quantifying context dependency of a plurality of sequence signals, the method comprising: (a) sequencing deoxyribonucleic acid (DNA) molecule(s) to provide a plurality of sequence signals, wherein said DNA molecule(s) comprise a known sequence; (b) aligning said plurality of sequence signals to a reference signal; and (c) quantifying said context dependency in said plurality of sequence signals aligned to said reference signal, based at least on said known sequence. In some embodiments, said aligning comprises analog signal processing. In some embodiments, the method further comprises: (d) sequencing a second set of DNA molecules comprising unknown sequences, thereby generating a second plurality of sequence signals; (e) aligning said second plurality of sequence signals to said reference signal; and (0determining homopolymer lengths of said second set of DNA molecules based at least on said plurality of sequence signals aligned to said reference signal and said quantified context dependency.

In some embodiments, said DNA molecule(s) is derived from a ribonucleic acid molecule(s). In some embodiments, said plurality of sequence signals is generated by massively parallel array sequencing. In some embodiments, said plurality of sequence signals is generated by flow sequencing. In some embodiments, quantifying said context dependency comprises establishing a context specific mapping between signal amplitudes and homopolymer length for each of a plurality of loci.

In another aspect, disclosed herein is a method for quantifying context dependency of a plurality of sequence signals, the method comprising: (a) sequencing deoxyribonucleic acid (DNA) molecule(s) to provide said plurality of sequence signals, wherein said DNA molecule(s) comprise a known sequence; (b) aligning said plurality of sequence signals to a reference signal; and (c) for each of a plurality of loci in said reference signal, determining an expected signal for said locus based at least on said plurality of sequence signals aligned to said reference signal and said known sequence. In some embodiments, said aligning comprises analog signal processing. In some embodiments, the method further comprises: (d) sequencing a second set of DNA molecules comprising unknown sequences, thereby generating a second plurality of sequence signals; (e) aligning said second plurality of sequence signals to said reference signal; and (0determining homopolymer lengths of said second set of DNA molecules based at least on said plurality of sequence signals aligned to said reference signal and said known sequence.

In some embodiments, said DNA molecule(s) is derived from a ribonucleic acid molecule(s). In some embodiments, said plurality of sequence signals is generated by massively parallel array sequencing. In some embodiments, said plurality of sequence signals is generated by flow sequencing.

In another aspect, disclosed herein is a method for processing a plurality of sequence signals, comprising: (a) sequencing a nucleic acid sample to provide said plurality of sequence signals; (b) aligning said plurality of sequence signals to a reference signal; (c) identifying a genomic locus comprising a homopolymer sequence based at least on said aligned sequence signals; (d) processing said plurality of sequence signals aligned to said reference signal to identify a presence and an estimated length of said homopolymer sequence; and (e) refining said estimated length using secondary assay data. In some embodiments, said aligning comprises analog signal processing. In some embodiments, said plurality of sequence signals is generated by massively parallel array sequencing. In some embodiments, said plurality of sequence signals is generated by flow sequencing.

In another aspect, disclosed herein is a system for sequencing a nucleic acid molecule, comprising: a database that stores a plurality of sequence signals generated upon sequencing said nucleic acid molecule; and one or more computer processors operatively coupled to said database, wherein said one or more computer processors are individual or collectively programmed to determine base calls of said nucleic acid molecule based at least in part on (i) said plurality of sequence signals and (ii) quantified context dependency for at least a portion of said plurality of sequence signals.

In another aspect, disclosed herein is a system for processing a plurality of sequence signals, comprising: a database that stores said plurality of sequence signals and a context dependency corresponding to said plurality of sequence signals; and one or more computer processors coupled to said database, wherein said one or more computer processors are individually or collectively programmed to: for each of a plurality of loci, determine an expected signal for said locus based at least on (i) said plurality of sequence signals and/or a plurality of imputed sequences corresponding to said plurality of sequence signals, and (ii) a known sequence. In some embodiments, said context dependency comprises a context specific mapping between signal amplitudes and known sequences for each of a plurality of loci.

In another aspect, disclosed herein is a system for processing a plurality of sequence signals and imputed sequences, comprising: a database that stores said plurality of sequence signals and imputed sequences; and one or more computer processors coupled to said database, wherein said one or more computer processors are individually or collectively programmed to: (a) truncate each identified imputed homopolymer sequence of at least N bases of said plurality of imputed sequences to a homopolymer sequence of bases of length N, to yield one or more HpN truncated sequences; (b) align said one or more HpN truncated sequences to a truncated reference(s), which truncated reference(s) has been HpN truncated and thereby comprises one or more homopolymer sequences truncated to said length N; and (c) generate a consensus sequence from said one or more HpN truncated sequences aligned to said HpN truncated reference(s), which consensus sequence comprises a homopolymer sequence of said length N, based at least on said one or more HpN truncated sequences aligned to said reference(s) and/or sequence signals associated with said one or more HpN truncated sequences aligned to said HpN truncated reference(s).

In another aspect, disclosed herein is a system for quantifying context dependency of a plurality of sequence signals and imputed sequences, comprising: a database that stores said plurality of sequence signals and imputed sequences; and one or more computer processors coupled to said database, wherein said one or more computer processors are individually or collectively programmed to: (a) truncate each identified imputed homopolymer sequence of at least N bases of said plurality of imputed sequences to a homopolymer sequence of bases of length N, to yield one or more HpN truncated sequences; (b) align said one or more HpN truncated sequences to a truncated reference(s), which truncated reference(s) has been HpN truncated and thereby comprises one or more homopolymer sequences truncated to said length N; and (c) quantify said context dependency of associated sequence signals based at least on (i) said one or more HpN truncated sequences aligned to said HpN truncated reference(s) and/or sequence signals associated with said one or more HpN truncated sequences aligned to said HpN truncated reference(s), and (ii) said known sequence. In some embodiments, said database stores training data, which training data comprises reference-aligned signals or context dependencies quantified from reference-aligned signals.

In another aspect, disclosed herein is a system for quantifying context dependency of a plurality of sequence signals and imputed sequences, comprising: a database that stores said plurality of sequence signals and imputed sequences; and one or more computer processors coupled to said database, wherein said one or more computer processors are individually or collectively programmed to: (a) truncate each identified imputed homopolymer sequence of at least N bases of said plurality of imputed sequences to a homopolymer sequence of bases of length N, to yield one or more HpN truncated sequences; (b) align said one or more HpN truncated sequences to a truncated reference(s), which truncated reference(s) has been HpN truncated and thereby comprises one or more homopolymer sequences truncated to said length N; and (c) for each of a plurality of loci in said HpN truncated reference(s), determine an expected signal for said locus based at least on (i) said one or more HpN truncated sequences aligned to said HpN truncated reference(s) and/or sequence signals associated with said one or more HpN truncated sequences aligned to said HpN truncated reference(s), and (ii) said known sequence.

In another aspect, disclosed herein is a system for processing a plurality of sequence signals and imputed sequences, comprising: a database that stores said plurality of sequence signals and imputed sequences; and one or more computer processors coupled to said database, wherein said one or more computer processors are individually or collectively programmed to: (a) process said plurality of sequence signals and imputed sequences to determine a set of one or more sequences comprising homopolymer sequences; (b) process said plurality of sequence signals and imputed sequences to identify a presence and an estimated length of at least a portion of said homopolymer sequences; and (c) refine said estimated lengths using secondary assay data.

In another aspect, disclosed herein is a system for processing a plurality of sequence signals, comprising: a database that stores said plurality of sequence signals; and one or more computer processors coupled to said database, wherein said one or more computer processors are individually or collectively programmed to: (a) align said plurality of sequence signals to a reference signal; (b) identify a reference locus comprising a homopolymer sequence based at least on said aligned sequence signals; and (c) generate a consensus sequence from said plurality of sequence signals aligned to said reference signal, which consensus sequence comprises a homopolymer sequence of N bases, based at least on said identified reference locus, a length of said homopolymer sequence of said reference locus, and said reference signal.

In another aspect, disclosed herein is a system for quantifying context dependency of a plurality of sequence signals, comprising: a database that stores said plurality of sequence signals; and one or more computer processors coupled to said database, wherein said one or more computer processors are individually or collectively programmed to: (a) align said plurality of sequence signals to a reference signal; and (b) quantify said context dependency in said plurality of sequence read signals aligned to said reference signal, based at least on a known sequence associated with said plurality of sequence signals.

In another aspect, disclosed herein is a system for quantifying context dependency of a plurality of sequence signals, comprising: a database that stores said plurality of sequence signals; and one or more computer processors coupled to said database, wherein said one or more computer processors are individually or collectively programmed to: (a) align said plurality of sequence signals to a reference signal; and (b) for each of a plurality of loci in said reference signal, determine an expected signal for said locus based at least on said plurality of sequence signals aligned to said reference signal and a known sequence associated with said plurality of sequence signals.

In another aspect, disclosed herein is a system for processing a plurality of sequence signals, comprising: a database that stores said plurality of sequence signals; and one or more computer processors coupled to said database, wherein said one or more computer processors are individually or collectively programmed to: (a) align said plurality of sequence signals to a reference signal; (b) identify a genomic locus comprising a homopolymer sequence based at least on said aligned sequence signals; (c) process said plurality of sequence signals aligned to said reference signal to identify a presence and an estimated length of said homopolymer sequence; and (d) refine said estimated length using secondary assay data.

Another aspect of the present disclosure provides a non-transitory computer readable medium comprising machine executable code that, upon execution by one or more computer processors, implements any of the methods above or elsewhere herein.

Another aspect of the present disclosure provides a system comprising one or more computer processors and computer memory coupled thereto. The computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein.

Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

