SYSTEMS AND METHODS FOR SAMPLE PREPARATION FOR SEQUENCING
The present disclosure provides compositions, methods, and systems for preparing nucleic acid samples for sequencing, including attaching adapters to template inserts, attaching template nucleic acid molecules to a support, enrichment. Further provided herein are methods of error correction by preserving both strands of a template nucleic acid molecule during amplification.
1 .- 89 . (canceled)
90 . A method for sequencing error correction by preserving sequences of both strands of a nucleic acid molecule during amplification, comprising:
(a) providing a substrate comprising a plurality of individually addressable locations, wherein a support is immobilized at an individually addressable location of the plurality of individually addressable locations, wherein the support comprises an amplified cluster of a plurality of nucleic acid molecules attached thereto wherein the plurality of nucleic acid molecules is derived from a double-stranded nucleic acid molecule which comprises a first strand and a second strand, wherein a first subset of the plurality of nucleic acid molecules each comprises a copy of at least a portion of the first strand and a second subset of the plurality of nucleic acid molecules each comprises a reverse complement copy of at least a portion of the second strand;
(b) sequencing the plurality of nucleic acid molecules in the amplified cluster by performing a plurality of flow steps and collecting a plurality of sequencing signals from the individually addressable location;
(c) identifying from the plurality of sequencing signals a phasing event at a flow step of the plurality of flow steps; and
(d) generating a sequencing read for the double-stranded nucleic acid molecule, wherein the sequencing read does not include a base call at a locus corresponding to the flow step where the phasing event was identified.
91 . The method of claim 90 , wherein the phasing event is attributed to an artificial base mismatch error at a locus in the double-stranded nucleic acid molecule.
92 . The method of claim 90 , wherein the phasing event at the flow step is identified by determining a read quality metric for each flow step of the plurality of flow steps, and determining that a given read quality metric at the flow step exceeds a predetermined threshold.
93 . The method of claim 90 , wherein the phasing event at the flow step is identified by determining a read quality metric for each flow step of the plurality of flow steps, and determining that a moving average of read quality metrics at or near the flow step exceeds a predetermined threshold.
94 . The method of claim 90 , further comprising generating a first sequencing read for the double-stranded nucleic acid molecule using the plurality of sequencing signals and trimming the first sequencing read based on the phasing event at the flow step to generate the sequencing read.
95 . The method of claim 90 , further comprising loading the support onto the substrate to immobilize the substrate at the individually addressable location.
96 . The method of claim 90 , wherein the support is a bead.
97 . The method of claim 90 , wherein the sequencing of (b) comprises providing nucleotide reagents according to a flow order comprising single base flows, wherein a single base flow comprises nucleotide reagents of a single base type.
98 . The method of claim 90 , wherein a given flow step of the plurality of flow steps comprises providing non-terminated nucleotides.
99 . The method of claim 90 , wherein a given flow step of the plurality of flow steps comprises providing a mixture of labeled and unlabeled nucleotides.
100 . The method of claim 90 , further comprising, upon detecting the phasing event at the flow step, ignoring or removing sequencing signals collected at the flow step and downstream of the flow step from base calling to generate the sequencing read.
101 . The method of claim 90 , further comprising identifying an error single nucleotide polymorphism (SNP) from the phasing event at the flow step and filtering the error SNP from SNP calling.
102 . The method of claim 90 , wherein a ratio of the first subset to the second subset is at least 0.2.
103 . The method of claim 102 , wherein the ratio is at least 0.4.
104 . The method of claim 103 , wherein the ratio is at least 0.6.
105 . A method for sequencing error correction by preserving sequences of both strands of a nucleic acid molecule during amplification, comprising:
(a) amplifying a double-stranded nucleic acid molecule, comprising a first strand and a second strand, to provide an amplified cluster of a plurality of nucleic acid molecules attached to a support, wherein a first subset of the plurality of nucleic acid molecules each comprises a copy of at least a portion of the first strand and a second subset of the plurality of nucleic acid molecules each comprises a reverse complement copy of at least a portion of the second strand;
(b) sequencing the plurality of nucleic acid molecules in the amplified cluster by performing a plurality of flow steps and collecting a plurality of sequencing signals;
(c) identifying from the plurality of sequencing signals a phasing event at a flow step of the plurality of flow steps; and
(d) generating a sequencing read for the double-stranded nucleic acid molecule, wherein the sequencing read does not include a base call at a locus corresponding to the flow step where the phasing event was identified.
106 . The method of claim 105 , further comprising generating a first sequencing read for the double-stranded nucleic acid molecule using the plurality of sequencing signals and trimming the first sequencing read based on the phasing event to generate the sequencing read.
107 . The method of claim 105 , further comprising, upon detecting the phasing event at the flow step, ignoring or removing sequencing signals collected at the flow step and downstream of the flow step from base calling to generate the sequencing read.
108 . The method of claim 105 , further comprising identifying an error single nucleotide polymorphism (SNP) from the phasing event and filtering the error SNP from SNP calling.
109 . The method of claim 105 , wherein a ratio of the first subset to the second subset is at least 0.4.