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US 11,597,967 B2Granted
Process for microsatellite instability detection
AssigneePersonal Genome Diagnostics Inc.
InventorsAndrew Georgiadis, Mark Sausen
Life of this patent
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Abstract
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.
Claims32
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.
12 more elements
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.
13 more elements
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.
13 more elements
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.
18 more elements
Dependencies are inferred from the claim text.