Methods and systems for sequence calling
The present disclosure provides methods and systems for accurate and efficient context-aware base calling of sequences. In an aspect, disclosed herein is a method for sequencing a nucleic acid molecule, comprising: (a) sequencing the nucleic acid molecule to generate a plurality of sequence signals; and (b) determining base calls of the nucleic acid molecule based at least in part on (i) the plurality of sequence signals and (ii) quantified context dependency for at least a portion of the plurality of sequence signals.
1. A method for processing a plurality of sequence signals, comprising:
(a) sequencing a nucleic acid to provide said plurality of sequence signals;
(b) aligning said plurality of sequence signals to a reference signal comprising a reference genome, thereby providing aligned sequence signals;
(c) identifying a reference locus comprising a reference homopolymer sequence based at least in part on said aligned sequence signals; and
(d) generating a consensus sequence from said aligned sequence signals, which consensus sequence comprises a consensus homopolymer sequence of N bases, based at least in part on said identified reference locus, a length of said reference homopolymer sequence, and said reference signal.
2. The method of claim 1 , further comprising determining a length estimation error of said consensus homopolymer sequence based at least in part on a distribution of signals or imputed homopolymer lengths of said plurality of said aligned sequence signals.
3. The method of claim 1 , further comprising, prior to (b), pre-processing said plurality of sequence signals to remove systematic error.
4. The method of claim 1 , wherein (a) comprises sequencing nucleic acids of a subject; and wherein said reference signal comprises a reference genome of a species of said subject.
5. The method of claim 4 , wherein a number of lengths computed or classified is restricted based at least in part on a ploidy of said species of said subject.
6. The method of claim 1 , wherein said plurality of sequence signals is generated at least in part by massively parallel array sequencing.
7. The method of claim 1 , wherein said plurality of sequence signals is generated at least in part by flow sequencing.
8. The method of claim 1 , wherein said reference signal comprises an analog reference signal, and wherein said aligning comprises performing analog signal processing.
9. The method of claim 1 , wherein said nucleic acid comprises deoxyribonucleic acid (DNA).
10. The method of claim 1 , wherein said nucleic acid comprises ribonucleic acid (RNA).
11. A method for quantifying context dependency of a plurality of sequence signals, the method comprising:
(a) sequencing a nucleic acid to provide said plurality of sequence signals, wherein said nucleic acid comprises a pre-determined sequence;
(b) aligning said plurality of sequence signals to a reference signal comprising a reference genome, thereby providing aligned sequence signals; and
(c) quantifying said context dependency in said aligned sequence signals, based at least in part on said pre-determined sequence.
12. The method of claim 11 , wherein said reference signal comprises an analog reference signal, and wherein said aligning comprises performing analog signal processing.
13. The method of claim 11 , further comprising:
(d) sequencing a test nucleic acid comprising a test sequence, thereby generating a second plurality of sequence signals;
(e) aligning said second plurality of sequence signals to said reference signal, thereby providing second aligned sequence signals; and
(f) determining homopolymer lengths of said test nucleic acid based at least in part on said second aligned sequence signals and said quantified context dependency.
14. The method of claim 11 , wherein said nucleic acid comprises a deoxyribonucleic acid (DNA) molecule.
15. The method of claim 11 , wherein (a) comprises sequencing nucleic acids of a subject; and wherein said reference signal comprises a reference genome of a species of said subject.
16. The method of claim 11 , wherein said nucleic acid comprises a ribonucleic acid (RNA) molecule.
17. The method of claim 11 , wherein said plurality of sequence signals is generated at least in part by massively parallel array sequencing.
18. The method of claim 11 , wherein said plurality of sequence signals is generated at least in part by flow sequencing.
19. The method of claim 11 , wherein quantifying said context dependency comprises establishing a context-specific mapping between signal amplitudes and homopolymer lengths for each locus of a plurality of loci.
20. The method of claim 11 , further comprising, for each of a plurality of loci in said reference signal, determining an expected signal for said locus based at least in part on said aligned sequence signals and said pre-determined sequence.