Method for sequencing a direct repeat

Abstract

Described herein is a method of sequencing a template that comprises a direct repeat, comprising: (a) in the same reaction, hybridizing a primer to a first site that is upstream of the first repeat sequence and hybridizing a primer to a second site that is upstream of the second repeat sequence, wherein the first and second sites are: (i) upstream of the first and second repeat sequences, respectively, and (ii) equidistant from the first and second repeat sequences; and (b) subjecting the hybridization product of (a) to a sequencing-by-synthesis sequencing reaction to produce a sequence read that comprises a combination of the first and second repeat sequences.

1 . A method of sequencing a template that comprises a first repeat sequence and a second repeat sequence, wherein the first and second repeat sequences are in a direct repeat and either identical or nearly identical, comprising:
 (a) in the same reaction, hybridizing a primer to a first site that is upstream of the first repeat sequence and hybridizing a primer to a second site that is upstream of the second repeat sequence, wherein the first and second sites are:
 (i) upstream of the first and second repeat sequences, respectively, and 
 (ii) equidistant from the first and second repeat sequences; and 
   (b) subjecting the hybridization product of (a) to a sequencing-by-synthesis sequencing reaction to produce a sequence read that comprises a combination of the first and second repeat sequences.   
     
     
         2 . The method of  claim 1 , wherein within each template the first repeat sequence and the second repeat sequence are amplified from opposite strands of a double-stranded fragment of DNA and are identical except for positions that correspond to damaged nucleotides in the double-stranded fragment of DNA or errors that occur during amplification. 
     
     
         3 . The method of  claim 2 , wherein the double-stranded fragment of DNA is genomic DNA. 
     
     
         4 . The method of  claim 3 , wherein the genomic DNA is eukaryotic genomic DNA. 
     
     
         5 . The method of  claim 3 , wherein the genomic DNA is isolated from a tissue biopsy. 
     
     
         6 . The method of  claim 3 , wherein the genomic DNA is cell-free DNA (cfDNA). 
     
     
         7 . The method of  claim 3 , wherein the genomic DNA is microbial genomic DNA. 
     
     
         8 . The method of  claim 3 , wherein the genomic DNA is viral genomic DNA. 
     
     
         9 . The method of  claim 1 , wherein the sequence read of (b) comprises, for each position of the sequence read, a quality score indicating the reliability of the base(s) called at that position. 
     
     
         10 . The method of  claim 9 , wherein a position in the sequence read that is uncalled or associated with a low-quality score indicates that first and second repeat sequences differ at a nucleotide that corresponds to that position. 
     
     
         11 . The method of  claim 10 , further comprising analyzing primary sequencing data for a position that has a low-quality score to determine the identities of the nucleotides at that position in the first and second repeats. 
     
     
         12 . The method of  claim 1 , wherein step (b) comprises:
 (i) reading a combination of signals obtained by simultaneous extension of the first and second primers to produce primary sequencing data;   (ii) processing the primary sequencing data using a base-calling algorithm to produce a sequence read composed of a sequence of base calls, each base call associated with a quality score indicating the reliability of the base call; and   (iii) outputting the sequence read based on (ii).   
     
     
         13 . The method of  claim 1 , wherein the sequencing-by-synthesis of step (b) comprises simultaneously extending the first and second primers in the presence of reversible chain terminators. 
     
     
         14 . The method of  claim 1 , wherein the first and second sites in the template are the same sequence. 
     
     
         15 . The method of  claim 1 , wherein the first and second sites in the template are different sequences. 
     
     
         16 . The method of  claim 1 , wherein the template comprises:
 (i) a first calibrator sequence that is present between the first site and the first repeat; and   (ii) a second calibrator sequence that is present between the second site and the second repeat, wherein the first and second calibrator sequences are the same length and have a different sequence; and   the sequence read of step (b) includes positions that correspond to the first and second calibrator sequences.   
     
     
         17 . The method of  claim 16 , further comprising analyzing the signals corresponding to the first and second calibrator sequences to determine how many strands of the first and second repeats are sequenced in the reaction. 
     
     
         18 . The method of  claim 17 , further comprising analyzing the signals corresponding to the first and second calibrator sequences to determine if a sufficient number of molecules have been sequenced. 
     
     
         19 . The method of  claim 1 , wherein first and second repeats are less than 2,000 nucleotides in length. 
     
     
         20 . The method of  claim 1 , wherein the method is done by:
 amplifying the template on a substrate by bridge PCR to produce a colony that comprises copies of the template;   hybridizing one or more primers to the colony, wherein a primer hybridizes to a first site that is upstream of the first repeat sequence and a primer hybridizes to a second site that is upstream of the second repeat sequence, wherein the first and second sites are: upstream of the first and second repeat sequences, respectively, and equidistant from the first and second repeat sequences; and   obtaining the sequence of the template by a sequencing-by-synthesis sequencing reaction to produce a sequence read that comprises a combination of the first and second repeat sequences.