NON-UNIQUE BARCODES IN A GENOTYPING ASSAY
The present disclosure involves ctDNA assays that interrogate many regions from a single sample with high precision and accuracy, while evaluating multiple forms of cancer-related genomic alterations including sequence mutations and structural alterations. The disclosure provides simplified yet robust methods that achieve high sensitivity and specificity by analyzing cancer genes using a limited pool of non-unique barcodes in combination with endogenous barcodes. Samples are captured and sequenced using high coverage next-generation sequencing to allow tumor-specific somatic mutations, amplifications, and translocations to be identified.
1 . A method for analyzing nucleic acids, the method comprising:
obtaining a sample comprising nucleic acid fragments;
introducing sets of non-unique barcodes to the fragments to generate a genomic library;
sequencing the fragments to produce sequence reads;
aligning the sequence reads;
identifying genomic positions of fragment ends; and
identifying a mutation that is present in multiple molecules as determined by a combination of non-unique barcodes and genomic position of fragment ends.
2 . The method of claim 1 , wherein the obtaining step comprises obtaining a plasma sample, and extracting nucleic acids.
3 . The method of claim 1 , wherein introducing sets of non-unique barcodes comprises end repair, A-tailing, and adapter ligation.
4 . The method of claim 1 , wherein the sets of non-unique barcodes consist of eight sets of non-unique barcodes.
5 . The method of claim 1 , wherein identifying genomic positions of fragment ends comprises hybrid capture or whole genome sequencing.
6 . The method of claim 1 , wherein genomic positions of fragment ends comprise endogenous barcodes.
7 . The method of claim 5 , wherein hybrid capture involves a panel of well-characterized cancer genes.
8 . The method of claim 7 , wherein the cancer genes include ABL1, AKT1, ALK, APC, AR, ATM, BCR, BRAF, CDH1, CDK4, CDK6, CDKN2A, CSF1R, CTNNB1, DNMT3A, EGFR, ERBB2, ERBB4, ESR1, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MAP2K1, MET, MLH1, MPL, MYC, NPM1, NRAS, NTRK1, PDGFRA, PDGFRB, PIK3CA, PIK3R1, PTEN, PTPN11, RARA, RB1, RET, ROS1, SMAD4, SMARCB1, SMO, SRC, STK11, TERT, TP53, and VHL.
9 . The method of claim 1 , wherein sequencing comprises single-end or paired-end sequencing.
10 . The method of claim 1 , wherein sequencing comprises redundant sequencing.
11 . The method of claim 10 , further comprising using the redundant sequence reads to determine a consensus sequence.
12 . The method of claim 10 , wherein redundant sequencing is performed at a depth of 10×.
13 . The method of claim 1 , wherein a mutation detected in a DNA molecule based on using non-unique barcodes and genomic positions of fragment ends that are identical across a predefined percentage of redundant sequence reads of the DNA molecule.
14 . The method of claim 13 , wherein the predefined percentage is 90%.
15 . The method of claim 1 , wherein nucleic acid comprises cell-free DNA, circulating tumor DNA, tumor-derived DNA, or RNA.
16 . The method of claim 1 , wherein the barcodes comprise sequencing adapters.
17 . A method for molecular barcoding, the method comprising:
obtaining a sample comprising nucleic acid fragments;
providing a plurality of sets of non-unique barcodes; and
tagging the nucleic acid fragments with the barcodes to generate a genomic library;
wherein each nucleic acid fragment is tagged with a same barcode as another different nucleic acid fragment in the genomic library.
18 . The method of claim 17 , wherein the plurality of sets is comprised of twenty or fewer unique barcodes.
19 . The method of claim 17 , wherein the plurality of sets is comprised of ten or fewer unique barcodes.
20 . The method of claim 17 , further comprising identifying genomic positions of fragment ends.
21 . The method of claim 17 , further comprising redundantly sequencing the genomic library to produce a plurality of redundant sequence reads of each nucleic acid fragment.
22 . The method of claim 21 , further comprising reconciling the redundant sequence reads of similarly-tagged nucleic acid fragments.
23 . The method of claim 22 , further comprising aligning the reconciled sequence reads to a reference to determine a consensus sequence.