Off-target capture reduction in sequencing techniques

Abstract

Presented herein are methods and compositions for enhancing specific enrichment of target sequences in a nucleic acid library. Off-target hybridization probes may be used to reduce binding and/or capture of off-target regions of a nucleic acid library in a targeted sequencing workflow. The off-target hybridization probes may be specific for locations known to generate off-target sequencing reads for a particular set of hybridization probes.

1 - 31 . (canceled) 
     
     
         32 . A sequencing kit for reducing off-target capture in a targeted sequencing reaction, comprising:
 a set of off-target hybridization probes that specifically bind to a plurality of off-target sequences present in a nucleic acid library generated from a sample, the nucleic acid library comprising a plurality of nucleic acid fragments; and   a set of target-specific hybridization probes that specifically bind to a plurality of target sequences present in the nucleic acid library.   
     
     
         33 . The kit of  claim 32 , wherein the off-target hybridization probes specifically bind to intronic sequences and the target-specific hybridization probes specifically bind to exonic sequences. 
     
     
         34 . The kit of  claim 32 , wherein the set of off-target hybridization probes comprises a universal set that is configured to be used with samples from a particular species. 
     
     
         35 . The kit of  claim 32 , wherein the probes of the set of off-target hybridization probes are between 80-120 bases in length. 
     
     
         36 . The kit of  claim 32 , wherein the probes of the set of target-specific hybridization probes are between 80-120 bases in length. 
     
     
         37 . The kit of  claim 32 , wherein the set of off-target hybridization probes comprises probes specific for a host sequence of a host, wherein the sample is not a same species as the host. 
     
     
         38 . The kit of  claim 32 , wherein the set of off-target hybridization probes is specific for 5000 or fewer different off-target sequences. 
     
     
         39 . The kit of  claim 32 , wherein the set of target-specific hybridization probes is specific for 10,000 or more different target sequences. 
     
     
         40 . The kit of  claim 32 , wherein the probes of the set of off-target hybridization probes and/or the set of target-specific hybridization probes comprise an affinity binding molecule of a binding pair. 
     
     
         41 . The kit of  claim 40 , wherein the probes of the set of off-target hybridization probes comprise the affinity binding molecule of the binding pair and the set of target-specific hybridization probes do not comprise the affinity binding molecule of the binding pair. 
     
     
         42 . The kit of  claim 40 , wherein the affinity binding molecule comprises biotin. 
     
     
         43 . A device for identifying probes for off-target sequence capture in a targeted sequencing reaction; the device comprising:
 a sample processing device configured to receive a reference group of target-specific and off-target nucleic acid fragments of a reference nucleic acid library generated from a reference sample, the reference nucleic acid library comprising a plurality of nucleic acid fragments, wherein the reference group of target-specific and off-target nucleic acid fragments are separated from the reference nucleic acid library based on binding to a set of target-specific hybridization probes;   an imager configured to image a substrate loaded with the reference group of target-specific and off-target nucleic acid fragments to generate sequencing data; and   a sequence analysis device configured to:
 receive the sequencing data to generate reference sequencing data; 
 identify off-target sequences in the reference sequencing data; and 
 identify a set of off-target hybridization probes based on the identified off-target sequences. 
   
     
     
         44 . The device of  claim 43 , wherein the sequence analysis device is configured to identify the set of off-target hybridization probes by ranking a prevalence of a plurality of off-target sequences in the sequencing data and selecting a plurality of highest prevalence off-target sequences to design the off-target hybridization probes such that the off-target hybridization probes are specific for the highest prevalence off-target sequences. 
     
     
         45 . The device of  claim 44 , wherein selecting the plurality of highest prevalence off-target sequences comprises selecting a predetermined number of off-target sequences according to the ranking. 
     
     
         46 . The device of  claim 45 , wherein the predetermined number is 5000 or fewer different off-target sequences. 
     
     
         47 . The device of  claim 44 , wherein selecting the plurality of highest prevalence off-target sequences comprises selecting a subset of off-target sequences associated with at least 50% of off-target sequence reads in the reference sequencing data. 
     
     
         48 . The device of  claim 43 , wherein the target-specific hybridization probes are specific for 10,000 or more different target sequences. 
     
     
         49 . The device of  claim 43 , wherein the sequence analysis device is configured to communicate the identified set of off-target hybridization probes to a synthesis facility. 
     
     
         50 . The device of  claim 43 , wherein the sequence analysis device is configured to generate instructions to synthesize the identified set of off-target hybridization probes. 
     
     
         51 . The device of  claim 43 , wherein the set of target-specific hybridization probes is a user-defined custom set of target-specific hybridization probes. 
     
     
         52 . The device of  claim 43 , wherein the sequence analysis device is configured to provide an estimated reduction in sequencing cost associated with using the off-target hybridization probes with the reference sample, wherein the estimated reduction in sequencing cost is based on a reduction in off-target sequences.