Methods of analyzing cell free nucleic acids and applications thereof
Processes and materials to detect neoplasms from a biopsy are described. Processes and materials to build a sequencing library are described. Processes and material to perform targeted sequencing are described. Processes and materials to mitigate confounding sources are described. Cell-free nucleic acids can be sequenced and the sequencing result can be utilized to detect sequences derived from a neoplasm.
1 . A method to prepare a DNA library for sequencing, the method comprising:
ligating onto a plurality of nucleic acid segments pairs of partial Y-adapters to flank each of the plurality of nucleic acid segments by a pair of partial Y-adapters, thereby producing a plurality of ligation products,
wherein each of the plurality of nucleic acid segments is DNA, and wherein the plurality of nucleic acid segments is obtained or derived from a biological sample,
wherein each of the pair of partial Y-adapters comprises an error-correcting unique identifier flanked by a 1 bp offset sequence and a 0-3 bp stagger sequence, and sequences for a primer to anneal in a grafting polymerase chain reaction, and
wherein the pair of error-correcting unique identifiers on each of the plurality of nucleic acid segments collectively provides a unique identification of the nucleic acid segments against other nucleic acid segments in the plurality of nucleic acid segments; and
grafting onto each of the plurality of ligation products a pair of error-correcting dual index sample barcodes to flank the ligation product by the error-correcting dual index sample barcodes, wherein the error-correcting dual index sample barcodes collectively provide a unique identification of the biological sample.
2 . The method of claim 1 , wherein the nucleic acid segment is complementary DNA (cDNA).
3 . The method of claim 1 , wherein the biological sample comprises cell-free DNA.
4 . The method of claim 1 , wherein the error-correcting dual index sample barcodes collectively provide the unique identification of the biological sample against other biological samples represented in the DNA library.
5 . The method of claim 1 , wherein the pair of error-correcting unique identifiers are each at least 3 bp in length.
6 . The method of claim 5 , wherein the error-correcting unique identifiers are each from 3 to 8 base pairs in length.
7 . The method of claim 1 , wherein the error-correcting unique identifiers are each 6 base pairs in length.
8 . The method of claim 1 , wherein the pair of error-correcting dual index sample barcodes each are 8 base pairs in length.
9 . The method of claim 1 , wherein the grafting comprises performing a polymerase chain reaction.
10 . The method of claim 1 , wherein the biological sample is a blood sample.
11 . The method of claim 1 , wherein the biological sample is a plasma sample.
12 . The method of claim 1 , wherein each of the pair of partial Y-adapters further comprises an overhang.
13 . The method of claim 12 , wherein the overhang comprises a T overhang.
14 . The method of claim 1 , further comprising after the ligating, performing a bead cleanup on the plurality of ligation products.
15 . The method of claim 1 , further comprising after the grafting, performing a sequencing reaction to generate a plurality of sequencing reads.
16 . The method of claim 1 , further comprising, before the ligating, performing an enrichment reaction on the plurality of nucleic acid segments.
17 . The method of claim 16 , wherein the enrichment reaction comprises use of a bait set to enrich for specific sequences of interest.