System and methods for detecting genetic variation

Abstract

The invention provides methods, apparatuses, and compositions for high-throughput amplification sequencing of specific target sequences in one or more samples. In some aspects, barcode-tagged polynucleotides are sequenced simultaneously and sample sources are identified on the basis of barcode sequences. In some aspects, sequencing data are used to determine one or more genotypes at one or more loci comprising a causal genetic variant. In some aspects, systems and methods of detecting genetic variation are provided.

1 .- 60 . (canceled) 
     
     
         61 . A method of enriching a plurality of different target polynucleotides in a sample comprising:
 (a) joining an adapter oligonucleotide to each of the target polynucleotides, wherein the adapter oligonucleotide comprises sequence Y;   (b) hybridizing a plurality of different oligonucleotide primers to the adapted target polynucleotides, wherein each oligonucleotide primer comprises sequence Z and sequence W;   wherein sequence Z is common among all oligonucleotide primers; and further wherein sequence W is different for each different oligonucleotide primer, is positioned at the 3′ end of each oligonucleotide primer, and is complementary to a sequence comprising a causal genetic variant or a sequence within 200 nucleotides of a causal genetic variant;   (c) in an extension reaction, extending the oligonucleotide primers along the adapted target polynucleotides to produce extended primers comprising sequence Z and sequence Y′, wherein sequence Y′ is complementary to sequence Y; and   (d) exponentially amplifying the purified extension products using a pair of amplification primers comprising (i) a first amplification primer comprising sequence V and sequence Z, wherein sequence Z is positioned at the 3′ end of the first amplification primer; and (ii) a second amplification primer comprising sequence X and sequence Y, wherein sequence Y is positioned at the 3′ end of the second amplification primer.   
     
     
         62 . The method of  claim 61 , wherein sequences W, Y, and Z are different sequences and comprise 5 or more nucleotides each. 
     
     
         63 . The method of  claim 61 , wherein each oligonucleotide primer comprises a first binding partner. 
     
     
         64 . The method of  claim 63 , wherein the method further comprises, before step (d), exposing the extended primers to a solid surface comprising a second binding partner that binds to the first binding partner, thereby purifying the extended primers away from one or more components of the extension reaction. 
     
     
         65 . The method of  claim 61 , wherein the plurality of oligonucleotide primers comprises at least about 100 different oligonucleotide primers each comprising a different sequence W selected from the group consisting of SEQ ID NOs 22-121 
     
     
         66 . The method of  claim 61 , wherein the target polynucleotides comprise fragmented polynucleotides that have a median length between about 200 and about 1000 base pairs, and wherein the fragmented polynucleotides are treated to produce blunt ends or to have a defined overhang prior to step (a), such as an overhang consisting of an adenine. 
     
     
         67 . The method of  claim 61  further comprising sequencing the products of step (d), wherein the sequencing comprises amplifying the products of step (d) by bridge amplification with bound oligonucleotides attached to a solid support to produce double-stranded bridge polynucleotides; cleaving one strand of a bridge polynucleotide at a cleavage site in a bound oligonucleotide; denaturing the cleaved bridge polynucleotide to produce a free single-stranded polynucleotide comprising a target sequence attached to the solid support; and sequencing the target sequence by extending a sequencing primer hybridized to at least a portion of one or more sequences added during one or more of steps (a), (c), or (d). 
     
     
         68 . The method of  claim 67 , wherein the sequencing comprises amplifying the products of step (d) by extension of a bound primer on a solid support to produce bound templates, hybridizing a sequencing primer to a bound template, extending the sequencing primer, and identifying nucleotides added by extension of the sequencing primer 
     
     
         69 . The method of  claim 61 , wherein the plurality of different oligonucleotide primers further comprises additional oligonucleotide primers comprising sequence Z and sequence W, wherein sequence W is different for each different additional oligonucleotide primer, is at the 3′ end of each additional oligonucleotide primer, and is complementary to a sequence comprising a non-subject sequence or a sequence within 200 nucleotides of a non-subject sequence. 
     
     
         70 . A method of using a gene sequencing device to detect genetic variation in a subject's genome comprising:
 (a) providing the device a plurality of clusters of polynucleotides, wherein (i) each cluster comprises multiple copies of a nucleic acid duplex attached to a support; (ii) each duplex in a cluster comprises a first molecule comprising sequences A-B-G′-D′-C′ from 5′ to 3′ and a second molecule comprising sequences C-D-G-B′-A′ from 5′ to 3′; (iii) sequence A′ is complementary to sequence A, sequence B′ is complementary to sequence B, sequence C′ is complementary to sequence C, sequence D′ is complementary to sequence D, and sequence G′ is complementary to sequence G; (iv) sequence G is a portion of a target polynucleotide sequence from a subject and is different for each cluster comprising a different sequence B; (v) sequence B′ is located 3′ with respect to sequence G in the corresponding target polynucleotide sequence; and (vi) each first molecule comprises a barcode sequence;   (b) having the device sequence G′ by extension of a first primer comprising sequence D to produce an R1 sequence for each cluster;   (c) having the device sequence B′ by extension of a second primer comprising sequence A to produce an R2 sequence for each cluster;   (d) having the device the barcode sequence by extension of a third primer to sequence C′ to produce a barcode sequence for each cluster;   (e) having the device a portion of an R1 sequence for a cluster when the portion of R1 sequence to be deleted is identical to at least a portion of sequence B′ for that cluster and sequence G is shorter than the R1 sequence for that cluster;   (f) having the device perform an alignment of R1 and R2 sequences to a reference sequence; and   (g) outputting the presence or absence of a sequence variation identified by step (f);   wherein steps (b) to (d) may be performed in any order.   
     
     
         71 . The method of  claim 70 , wherein the first reference sequence comprises a reference genome. 
     
     
         72 . The method of  claim 70 , wherein the second reference sequence consists of every sequence B for every different target polynucleotide. 
     
     
         73 . The method of  claim 70 , wherein in step (f) the R1 and the R2 sequences are aligned to the same reference sequence. 
     
     
         74 . The method of  claim 70 , further comprising discarding an R1 sequence that aligns to a first position in the reference sequence that is more than 10,000 base pairs away from a second position in the reference sequence to which the R2 sequence for the same cluster aligns. 
     
     
         75 . The method of  claim 70 , wherein the outputting step comprises a computer file containing a sequence of the nucleic acid aligned to a sequence of the reference genome. 
     
     
         76 . The method of  claim 70 , wherein the outputting step comprises updating a display and a database. 
     
     
         77 . The method of  claim 70 , wherein each barcode differs from every other barcode in a plurality of different barcodes analyzed in parallel. 
     
     
         78 . The method of  claim 70 , wherein the barcode sequence is located 5′ from sequence D′. 
     
     
         79 . The method of  claim 70 , further comprising grouping sequences from the clusters based on the barcode sequences, and discarding all but one of a plurality of R1 sequences having the same sequence and alignment within a barcode sequence grouping. 
     
     
         80 . The method of  claim 70 , further comprising producing a single consensus sequence, wherein the consensus sequence identifies an insertion, a deletion, or an insertion and a deletion in a target polynucleotide with an accuracy of at least about 90%.