Process for microsatellite instability detection
The invention provides methods for determining the MSI status of a patient by liquid biopsy with sample preparation using hybrid capture and non-unique barcodes. In certain aspects, the invention provides a method of detecting microsatellite instability (MSI). The method includes obtaining cell-free DNA (cfDNA) from a sample of blood or plasma from a patient and sequencing portions of the cfDNA to obtain sequences of a plurality of tracts of nucleotide repeats in the cfDNA. A report is provided describing an MSI status in the patient when a distribution of lengths of the plurality of tracts has peaks that deviate significantly from peaks in a reference distribution.
1. A method of determining microsatellite instability (MSI) status comprising:
(a) capturing target cell free DNA (cfDNA) from a liquid sample from a patient, wherein the liquid sample is from blood, serum, plasma, urine, saliva or other biological fluid;
(b) attaching a plurality of sets of non-unique exogenous barcodes to the cfDNA so that each cfDNA molecule is attached to a non-unique exogenous barcode;
(c) amplifying the cfDNA from (b) to produce amplicons that include barcode information and copies of the cfDNA molecules;
(d) sequencing the amplicons to obtain sequences of a plurality of tracts of nucleotide repeats, wherein the obtained sequences include a plurality of sequence reads;
(e) identifying groups of sequence reads that originated from each unique cfDNA molecule by means of the barcode information and position or content of the sequence reads;
(f) performing a first alignment of the plurality of sequence reads from (e) with a reference sequence, wherein the reference sequence comprises a human genome;
(g) identifying a subset of the sequence reads aligning with microsatellite alleles in the reference sequence;
(h) performing a second alignment on the subset of sequence reads from (g) with their respective microsatellite alleles in the reference sequence to determine indel length for each sequence read within the subset;
(i) aggregating groups of sequence reads according to the first and second alignment;
(j) performing error correction using the non-unique exogenous barcodes on the aggregated groups of sequence reads from (i) which comprise at least 2 sequence reads and wherein more than 50% of those sequence reads comprise identical nucleotide repeat lengths;
(k) identifying local peaks from a length distribution plot of nucleotide repeat lengths from each aggregated group of corrected sequence reads from (j); and
(l) determining if the length of a microsatellite allele corresponding to a local peak from (k) is 3 or more base pairs shorter than the length of the corresponding microsatellite allele in the reference sequence,
wherein local peaks which are 3 or more base pairs shorter than the length of the corresponding microsatellite allele in the reference sequence are determined to have MSI;
thereby determining MSI status.
2. The method of claim 1 , wherein the reference sequence is from a matched normal DNA sample.
3. The method of claim 1 , wherein the microsatellite alleles include one or more of BAT25, BAT26, MONO-27, NR21, NR24, Penta C, and Penta D.
4. The method of claim 1 , wherein the microsatellite alleles include BAT25, BAT26, MONO-27, NR21, and NR24.
5. The method of claim 1 , further comprising recommending a treatment for the patient based on the MSI status.
6. The method of claim 5 , wherein the MSI status indicates that the patient is microsatellite instable and the treatment comprises an immune checkpoint inhibitor.
7. The method of claim 1 , further comprising administering an immune checkpoint inhibitor to the patient.
8. The method of claim 7 , wherein the immune checkpoint inhibitor comprises an antibody.
9. The method of claim 8 , wherein the antibody is selected from the group consisting of: an anti-PD-1 antibody; an anti-IDO antibody; an anti-CTLA-4 antibody; an anti-PD-L1 antibody; and an anti-LAG-3 antibody.
10. The method of claim 1 , wherein the sequencing is next-generation, short-read sequencing.
11. The method of claim 1 , wherein the liquid sample is from plasma.
12. The method of claim 1 , wherein the microsatellite alleles comprise a mononucleotide repeat.
13. The method of claim 1 , wherein the method is used in screening for Lynch syndrome.
14. The method of claim 1 , wherein the patient has a cancer selected from pancreatic, colon, gastric, endometrial, cholangiocarcinoma, breast, lung, head and neck, kidney, bladder, prostate cancer, or hematopoietic cancers.
15. A method for generating a personalized cancer treatment report for a patient having or suspected of having cancer, the method comprising:
(a) capturing target cell free DNA (cfDNA) from a liquid sample from a patient, wherein the liquid sample is from blood, serum, plasma, urine, saliva or other biological fluid;
(b) attaching a plurality of sets of non-unique exogenous barcodes to the cfDNA so that each cfDNA molecule is attached to a non-unique exogenous barcode;
(c) amplifying the cfDNA from (b) to produce amplicons that include barcode information and copies of the cfDNA molecules;
(d) sequencing the amplicons to obtain sequences of a plurality of tracts of nucleotide repeats, wherein the obtained sequences include a plurality of sequence reads;
(e) identifying groups of sequence reads that originated from each unique cfDNA molecule by means of the barcode information and position or content of the sequence reads;
(f) performing a first alignment of the plurality of sequence reads from (e) with a reference sequence, wherein the reference sequence comprises a human genome;
(g) identifying a subset of the sequence reads aligning with microsatellite alleles in the reference sequence;
(h) performing a secondary alignment on the subset of sequence reads from (g) with their respective microsatellite alleles in the reference sequence, to determine indel length for each sequence read within the subset;
(i) aggregating groups of sequence reads according to the first and second alignment;
(j) performing error correction using the non-unique exogenous barcodes on the aggregated groups of sequence reads from (i) which comprise at least 2 sequence reads and wherein more than 50% of those sequence reads comprise identical nucleotide repeat lengths;
(k) identifying local peaks from a length distribution plot of nucleotide repeat lengths from each aggregated group of corrected sequence reads from (j); and
(l) determining if the length of the microsatellite allele corresponding to a local peak from (k) is 3 or more base pairs shorter than the length of the corresponding microsatellite allele in the reference sequence,
wherein local peaks which are 3 or more base pairs shorter than the length of the corresponding microsatellite allele in the reference sequence are determined to have MSI; and
generating a personalized cancer treatment report.
16. The method of claim 15 , wherein the cancer treatment report comprises one or more of the following: (i) information on prognosis, resistance, or potential therapeutic options; (ii) information on the likely effectiveness of a therapeutic option; (iii) the acceptability of a therapeutic option, or the advisability of applying the therapeutic option to the subject; or (iv) information on the administration of a drug.
17. The method of claim 15 , wherein the patient has a cancer selected from pancreatic, colon, gastric, endometrial, cholangiocarcinoma, breast, lung, head and neck, kidney, bladder, prostate cancer, or hematopoietic cancers.
18. A method of determining the prognosis or therapeutic regimen for a patient having cancer comprising:
(a) capturing target cell free DNA (cfDNA) from a liquid sample from a patient, wherein the sample is from blood, serum, plasma, urine, saliva or other biological fluid;
(b) attaching a plurality of sets of non-unique exogenous barcodes to the cfDNA so that each cfDNA molecule is attached to a non-unique barcode;
(c) amplifying the cfDNA from (b) to produce amplicons that include barcode information and copies of the cfDNA molecules;
(d) sequencing the amplicons to obtain sequences of a plurality of tracts of nucleotide repeats, wherein the obtained sequences include a plurality of sequence reads;
(e) identifying groups of sequence reads that originated from each unique cfDNA molecule by means of the barcode information and position or content of the sequence reads;
(f) performing a first alignment of the plurality of sequence reads from (e) with a reference sequence, wherein the reference sequence comprises a human genome;
(g) identifying a subset of the sequence reads aligning with microsatellite alleles in the reference sequence;
(h) performing a secondary alignment on the subset of sequence reads from (g) with their respective microsatellite alleles in the reference sequence, to determine indel length for each sequence read within the subset;
(i) aggregating groups of sequence reads according to the first and second alignment;
(j) performing error correction using the non-unique exogenous barcodes on the aggregated groups of sequence reads from (i) which comprise at least 2 sequence reads and wherein more than 50% of those sequence reads comprise identical nucleotide repeat lengths;
(k) identifying local peaks from a length distribution plot of nucleotide repeat lengths from each aggregated group of corrected sequence reads from (j); and
(l) determining if the length of the microsatellite allele corresponding to a local peak from (k) is 3 or more base pairs shorter than the length of the corresponding microsatellite allele in the reference sequence,
wherein local peaks which are 3 or more base pairs shorter than the length of the corresponding microsatellite allele in the reference sequence are determined to have MSI; and
determining a prognosis or therapeutic regimen for the patient based on the MSI status for each microsatellite allele.
19. The method of claim 18 , wherein the reference sequence is from a matched normal DNA sample.
20. The method of claim 18 , wherein the microsatellite alleles include one or more of BAT25, BAT26, MONO-27, NR21, NR24, Penta C, and Penta D.
21. The method of claim 18 , wherein the microsatellite alleles include BAT25, BAT26, MONO-27, NR21, and NR24.
22. The method of claim 18 , further comprising administering an immune checkpoint inhibitor to the patient.
23. The method of claim 22 , wherein the immune checkpoint inhibitor is selected from the group consisting of: an anti-PD-1 antibody; an anti-IDO antibody; an anti-CTLA-4 antibody; an anti-PD-L1 antibody; and an anti-LAG-3 antibody.
24. The methods of any one of claim 1 , 15 or 18 , wherein the MSI status is determined within a specificity of >99% and a sensitivity of 75%.
25. The method of claim 24 , wherein the liquid sample has a tumor fraction of <5%.
26. The method of claim 24 , wherein the liquid sample has a tumor fraction of 0.5-7.9%.
27. The method of any one of claims 1 , 15 or 18 , wherein local peaks comprise:
(a) a greater number of sequences reads as compared to the number of sequence reads adjacent to the local peak at each individual length within ±2 bp in the length distribution plot,
(b) more than 3 distinct fragments having the same length, and
(c) more than 1% absolute coverage of a microsatellite sequence in the reference.
28. The methods of any one of claim 1 , 15 or 18 , wherein the reference genome is the human genome assembly hg19.
29. A method of determining microsatellite instability (MSI) status comprising:
(a) capturing target cell free DNA (cfDNA) from a liquid sample from a patient, wherein the liquid sample is from blood, serum, plasma, urine, saliva or other biological fluid;
(b) attaching a plurality of sets of non-unique exogenous barcodes to the cfDNA so that each cfDNA molecule is attached to a non-unique exogenous barcode;
(c) amplifying the fragments to produce amplicons that include barcode information and copies of the cfDNA molecules;
(d) sequencing the amplicons to obtain sequences of a plurality of tracts of nucleotide repeats, wherein the obtained sequences include a plurality of sequence reads;
(e) identifying groups of sequence reads that originated from each unique cfDNA molecule by means of the barcode information and position or content of the sequence reads;
(f) performing a first alignment of the plurality of sequence reads from (e) with a reference sequence, wherein the reference sequence comprises a human genome;
(g) identifying a subset of the sequence reads aligning with microsatellite alleles in the reference sequence, and which further comprise:
(i) a nucleotide repeat beginning >8 bp after the start of the sequence read and ending >8 bp from the end of the sequence read,
(ii) an insertion or deletion (indel) comprising 12 or less bases compared to the reference,
(iii) the absence of a single base change within the nucleotide repeat, and
(iv) a mapping score of 60, and
(v) 20 or less soft clipped bases;
(h) performing a secondary alignment on the subset of sequence reads from (g) with their respective microsatellite alleles in the reference sequence, to determine indel length for each sequence read within the subset;
(i) aggregating groups of sequence reads within the subset of sequence reads according to the first alignment from (f), the second alignment from (h), and alignment of non-unique exogenous barcodes;
(j) performing error correction using the non-unique exogenous barcodes on the aggregated groups of sequence reads from (i) which comprise at least 2 sequence reads and wherein more than 50% of those sequence reads comprise identical nucleotide repeat lengths;
(k) identifying local peaks from a length distribution plot of nucleotide repeat lengths from each aggregated group of corrected sequence reads from (j); and
(l) determining if the length of the microsatellite allele corresponding to a local peak from (k) is 3 or more base pairs shorter than the length of the corresponding microsatellite allele in the reference sequence,
wherein local peaks which are 3 or more base pairs shorter than the length of the corresponding microsatellite allele in the reference sequence are determined to have MSI,
wherein MSI status is determined within a specificity of >99% and a sensitivity of 75%, and
thereby determining microsatellite instability (MSI) status.
30. The method of claim 29 , wherein the liquid sample has a tumor fraction of <5%.
31. The method of claim 29 , wherein the liquid sample has a tumor fraction of 0.5-7.9%.
32. The method of claim 29 , wherein the human genome is the human genome assembly hg19.