Polynucleotide barcodes for long read sequencing

Abstract

Provided herein are methods of making, amplifying, and sequencing tagged nucleic acid complements, compositions including interposing oligonucleotide barcodes, and kits useful in obtaining long-range sequence data.

1 .- 18 . (canceled) 
     
     
         19 . A plurality of interposing oligonucleotide barcodes capable of hybridizing to a sample polynucleotide, the interposing oligonucleotide barcodes comprising from 5′ to 3′:
 i. a first hybridization pad complementary to a first sequence of the sample polynucleotide; 
 ii. a first stem region comprising a sequence common to the plurality of interposing oligonucleotide barcodes; 
 iii. a loop region comprising a barcode sequence, wherein the barcode sequence, alone or in combination with a sequence of one or both of (a) the sample polynucleotide, or (b) one or more additional barcode sequences, uniquely distinguishes the sample polynucleotide from other sample polynucleotides in the plurality; 
 iv. a second stem region comprising a sequence complementary to the first stem region, wherein the second stem region is capable of hybridizing to the first stem region under hybridization conditions; and 
 v. a second hybridization pad complementary to a second sequence of the sample polynucleotide. 
 
     
     
         20 . (canceled) 
     
     
         21 . The interposing oligonucleotide barcodes of  claim 19 , wherein each hybridization pad comprises a targeted primer sequence. 
     
     
         22 . (canceled) 
     
     
         23 . The interposing oligonucleotide barcodes of  claim 19 , wherein the barcode sequence comprises about 5 to about 20 nucleotides, or about 10 to about 20 nucleotides. 
     
     
         24 .- 30 . (canceled) 
     
     
         31 . The interposing oligonucleotide barcodes of  claim 19 , wherein the barcode sequence alone uniquely distinguishes the sample polynucleotide from other sample polynucleotides in the plurality. 
     
     
         32 . The interposing oligonucleotide barcodes of  claim 19 , wherein the barcode sequence in combination with a sequence of one or both of (a) the sample polynucleotide, or (b) one or more additional barcode sequences, uniquely distinguishes the sample polynucleotide from other sample polynucleotides in the plurality. 
     
     
         33 . The interposing oligonucleotide barcodes of  claim 19 , wherein the barcode sequence comprises about 5 to 15 nucleotides. 
     
     
         34 . The interposing oligonucleotide barcodes of  claim 19 , wherein each of the interposing oligonucleotide barcodes comprises a phosphorylated 5′ end. 
     
     
         35 . A composition comprising a sample polynucleotide hybridized to the plurality of oligonucleotides barcodes of  claim 19 . 
     
     
         36 . The composition of  claim 35 , wherein the second hybridization pad of each interposing oligonucleotide barcode is at least twice as long as the first hybridization pad of each interposing oligonucleotide barcode. 
     
     
         37 . The composition of  claim 35 , wherein the second hybridization pad of each interposing oligonucleotide barcode is about the same length as the first hybridization pad of each interposing oligonucleotide barcode. 
     
     
         38 . The composition of  claim 35 , wherein the first and second stem regions are complementary and wherein each stem region comprises a known sequence of about 5 to about 10 nucleotides. 
     
     
         39 . The composition of  claim 35 , wherein the loop region comprises about 5 to about 20 nucleotides, or about 10 to about 20 nucleotides. 
     
     
         40 . The composition of  claim 35 , wherein the sample polynucleotide comprises a gene or a gene fragment, wherein the gene or gene fragment is a cancer-associated gene or fragment thereof, T cell receptor (TCRs) gene or fragment thereof, or a B cell receptor (BCRs) gene, or fragment thereof. 
     
     
         41 . The composition of  claim 35 , wherein the sample polynucleotide comprises genomic DNA, complementary DNA (cDNA), cell-free DNA (cfDNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), cell-free RNA (cfRNA), or noncoding RNA (ncRNA). 
     
     
         42 . The composition of  claim 35 , wherein the sample polynucleotide is at least 1 kb in length. 
     
     
         43 . A polynucleotide comprising a plurality of units, wherein each unit comprises a portion of a genomic sequence and a sequence of an interposing oligonucleotide barcode, wherein each interposing oligonucleotide barcode comprises from 5′ to 3′:
 a. a first stem region comprising a sequence common to the plurality of units; 
 b. a loop region comprising a barcode sequence, wherein each barcode sequence in the polynucleotide is different; and 
 c. a second stem region comprising a sequence complementary to the first stem region, wherein the second stem region hybridizes to the first stem region during said hybridizing. 
 
     
     
         44 . A plurality of the polynucleotides of  claim 43 , wherein each polynucleotide in the plurality comprises a different combination of barcode sequences. 
     
     
         45 . A kit comprising the plurality of interposing oligonucleotides barcodes of  claim 19 . 
     
     
         46 . The kit of  claim 45 , further comprising a first terminal adapter and a second terminal adapter, wherein each terminal adapter comprises at least one hybridization pad, a barcode, and a primer binding site, and further wherein each terminal adapter does not comprise a loop region or a stem region. 
     
     
         47 . The kit of  claim 46 , wherein the first terminal adapter comprises, from 5′ to 3′, a primer binding sequence, a barcode, a first hybridization pad and a second hybridization pad complementary to the 3′ end of the sample polynucleotide, and wherein the second terminal adapter comprises, from 5′ to 3′, a first hybridization pad and a second hybridization pad, an index, and a primer binding sequence, wherein the first and the second hybridization pads anneal to the 5′ end of the sample polynucleotide.