Methods, systems, compositions, kits, apparatus and computer-readable media for molecular tagging
In some embodiments, the disclosure relates generally to methods, as well as related systems, compositions, kits, apparatuses and computer-readable media, comprising a multiplex molecular tagging procedure that employs a plurality of tags that are appended to a plurality of polynucleotides. The tags have characteristics, including a sequence, length and/or detectable moiety, or any other characteristic, that uniquely identifies the polynucleotide molecule to which it is appended, and permits tracking individual tagged molecules in a mixture of tagged molecules. For example, the tag having a unique tag sequence, can uniquely identify an individual polynucleotide to which it is appended, and distinguish the individual polynucleotide from other tagged polynucleotides in a mixture. In some embodiments, the multiplex molecular tagging procedure can be used for generating error-corrected sequencing data and for detecting a target polynucleotide which is present at low abundance in a nucleic acid sample.
1. A method for detecting a genetic variant in a nucleic acid sample, comprising:
a) appending individual polynucleotides of a plurality of polynucleotides of the nucleic acid sample with at least one oligonucleotide tag of a plurality of oligonucleotide tags to generate tagged polynucleotides, wherein the at least one oligonucleotide tag comprises a structure, N 1 N 2 N 3 X 1 X 2 X 3 M 4 M 5 M 6 Y 4 Y 5 Y 6
(i) wherein N represents a random tag sequence wherein each base position in the random tag sequence is independently selected from A, G, C or T;
(ii) wherein X 1 X 2 X 3 represents a first fixed tag sequence that is the same in all of the plurality of tags;
wherein M represents a random tag sequence wherein each base position in the random tag sequence is independently selected from A, G, C or T, wherein the random tag sequence M differs from the random tag sequence N; and
(iv) wherein Y 4 Y 5 Y 6 represents a second fixed tag sequence that is the same in all of the plurality of tags, and the second fixed tag sequence of Y 4 Y 5 Y 6 differs from the first fixed tag sequence of X 1 X 2 X 3 ;
b) amplifying the tagged polynucleotides to generate tagged amplicons;
c) sequencing the tagged amplicons to generate a plurality of candidate sequencing reads, wherein each candidate sequencing read includes at least a portion of a sequence corresponding to the polynucleotide and at least a portion of a sequence corresponding to the at least one oligonucleotide tag that is appended to the polynucleotide, wherein the candidate sequencing reads are stored in a memory in communication with a processor;
d) grouping the candidate sequencing reads into families of grouped candidate sequencing reads having a common tag sequence that is unique to a given family of candidate sequencing reads;
e) removing mistagged sequencing reads from the families of candidate sequencing reads to produce error-corrected families of sequencing reads, wherein a mistagged sequencing read has the common tag sequence for the given family but corresponds to a different polynucleotide than other candidate sequencing reads in the given family due to errors in the appending of step a); and
f) detecting a variant in a plurality of error-corrected families of sequencing reads.
2. The method of claim 1 , further comprising identifying an error in one or more of the candidate sequencing reads.
3. The method of claim 2 , wherein the step of identifying an error further includes applying a culling threshold to a number of nucleotides that differ between the candidate sequencing read and a reference sequence to identify a candidate sequencing read having an error.
4. The method of claim 3 , wherein the reference sequence is a tag-specific reference sequence.
5. The method of claim 3 , wherein the reference sequence is a polynucleotide-specific reference sequence.
6. The method of claim 1 , wherein the removing mistagged sequencing reads of step e) further includes comparing the candidate sequencing read in the given family to a polynucleotide-specific reference sequence to determine a number of nucleotides that differ between the candidate sequencing read and the polynucleotide-specific reference sequence.
7. The method of claim 6 , wherein the removing mistagged sequencing reads of step e) further includes applying a difference counting threshold to identify a mistagged sequencing read.
8. The method of claim 1 , wherein the removing mistagged sequencing reads of step e) includes comparing the candidate sequencing read to one or more other candidate sequencing reads in the given family to identify candidate sequencing reads having a common pattern of variants.
9. The method of claim 8 , wherein the removing mistagged sequencing reads of step e) further includes applying a pattern counting threshold to a number of candidate sequencing reads having the common pattern of variants to identify a group of mistagged sequencing reads.
10. The method of claim 1 , wherein the removing mistagged sequencing reads of step e) includes comparing the candidate sequencing reads in the given family to a polynucleotide-specific reference sequence to identify a candidate mistagged sequencing read.
11. The method of claim 10 , wherein the removing mistagged sequencing reads of step e) further includes comparing the candidate mistagged sequencing read to one or more other candidate mistagged sequencing reads in the given family to identify a common pattern of variants.
12. The method of claim 11 , wherein the removing mistagged sequencing reads of step e) further includes applying a pattern counting threshold to a number of candidate mistagged sequencing reads having the common pattern of variants to determine a group of mistagged sequencing reads.
13. The method of claim 1 , wherein the removing mistagged sequencing reads of step e) includes comparing the candidate sequencing read in the given family to a polynucleotide-specific reference sequence to identify a pattern of differences in a candidate mistagged sequencing read.
14. The method of claim 13 , wherein the removing mistagged sequencing reads of step e) further includes determining a number of matches for the pattern of differences in the candidate mistagged sequencing read compared to a pattern of expected differences between the polynucleotide-specific reference sequence and an expected sequence for a non-target polynucleotide.
15. The method of claim 14 , wherein the removing mistagged sequencing reads of step e) further includes applying a non-target pattern threshold to the number of matches to identify a mistagged sequencing read.
16. The method of claim 1 , wherein the detecting of step f) includes aligning the sequencing reads for the error-corrected family to a polynucleotide-specific reference sequence.
17. The method of claim 16 , wherein the detecting of step f) further includes counting a number of aligned sequencing reads having a particular base difference at a given position with respect to the polynucleotide-specific reference sequence.
18. The method of claim 17 , wherein the detecting of step f) further includes applying a family level threshold to the number of aligned sequencing reads to identify a family-based candidate variant.
19. The method of claim 1 , the detecting of step f) further includes counting a number of error-corrected families having a particular family-based candidate variant.
20. The method of claim 19 , the detecting of step f) further includes applying a multi-family threshold to the number of error-corrected families to identify the variant.