METHODS FOR TAGGING AND AMPLIFYING RNA TEMPLATE MOLECULES FOR PREPARING SEQUENCING LIBRARIES
First Claim
Patent Images
1. A method for preparing a sequencing library from a test sample comprising RNA, the method comprising the steps:
- (a) obtaining a test sample comprising RNA sequences, and purifying the RNA sequences from the test sample;
(b) synthesizing first complementary DNA (cDNA) strands based on the RNA sequences and C-tailing 3′
-end of cDNA strand;
(c) annealing a complementary template switching oligonucleotide to the C-tail of the cDNA and ligating the complementary template switching oligonucleotide to the 5′
-ends of the RNA sequences to produce RNA templates; and
(d) synthesizing a plurality of cDNA strands from the RNA templates using a strand-displacement reverse transcriptase.
2 Assignments
0 Petitions
Accused Products
Abstract
Aspects of the invention include methods for preparing sequencing libraries comprising a plurality of RNA molecules. In some embodiments, the subject methods comprise sequencing at least a portion of a sequencing library to obtain sequencing data or sequence reads from a test sample (e.g., a biological sample from a subject).
-
Citations
30 Claims
-
1. A method for preparing a sequencing library from a test sample comprising RNA, the method comprising the steps:
-
(a) obtaining a test sample comprising RNA sequences, and purifying the RNA sequences from the test sample; (b) synthesizing first complementary DNA (cDNA) strands based on the RNA sequences and C-tailing 3′
-end of cDNA strand;(c) annealing a complementary template switching oligonucleotide to the C-tail of the cDNA and ligating the complementary template switching oligonucleotide to the 5′
-ends of the RNA sequences to produce RNA templates; and(d) synthesizing a plurality of cDNA strands from the RNA templates using a strand-displacement reverse transcriptase. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 12, 16, 18, 19, 26)
-
-
2. A method for preparing a sequencing library from a test sample comprising RNA, the method comprising the steps:
-
(a) obtaining a test sample comprising one or more RNA sequences, and purifying the one or more RNA sequences from the test sample; (b) annealing a first RNA primer to the one or more RNA sequences; (c) extending the first RNA primer in a first nucleic acid extension reaction using reverse transcriptase, wherein the reverse transcriptase comprises reverse transcription and terminal transferase activities, to generate a plurality of DNA sequences complementary to the one or more RNA templates, and wherein the complementary DNA (cDNA) sequences further comprise a plurality of non-templated bases at the 3′
-end of the cDNA sequences;(d) annealing a complementary nucleic acid sequence to the non-templated bases at the 3′
-end of the cDNA sequence, wherein the complementary nucleic acid sequence further comprises a unique molecular identifier (UMI) or a unique sequence tag;(e) ligating the complementary nucleic acid sequence to the 5′
-end of the one or more RNA sequences to generate one or more RNA templates, wherein the one or more RNA templates comprise the original one or more RNA sequences covalently linked to the complementary nucleic acid sequence comprising the UMI or unique sequence tag;(f) annealing one or more second RNA primers to the one or more RNA template; and (g) extending the one or more second RNA primers in a second nucleic acid extension reaction using a strand-displacement reverse transcriptase to generate a plurality of DNA sequence complementary to the one or more RNA templates, wherein the plurality of complementary DNA (cDNA) sequences each comprise the complementary DNA sequence and a UMI or unique sequence tag. - View Dependent Claims (10, 11, 13, 14, 15, 17)
-
-
20. A method for preparing a sequencing library from a test sample comprising RNA, the method comprising the steps:
-
(a) obtaining a test sample comprising one or more RNA sequences, and purifying the one or more RNA sequences from the test sample; (b) annealing a first RNA primer to the one or more RNA sequences; (c) extending the first RNA primer in a first nucleic acid extension reaction using a reverse transcriptase, wherein the reverse transcriptase comprises reverse transcription and terminal transferase activities, to generate a plurality of DNA sequences complementary to the one or more RNA templates, wherein the terminal transferase activity adds a cytosine (C) tail to the 3′
-end of the complementary DNA (cDNA) sequences;(d) annealing a template switching oligonucleotide to the 3′
-cytosine tail of the cDNA sequence, wherein the template switching oligonucleotide sequence further comprises a unique molecular identifier (UMI) or a unique sequence tag;(e) ligating the template switching oligonucleotide to the 5′
-end of the one or more RNA sequences with T4 RNA ligase to generate one or more RNA templates, wherein the RNA templates comprise the original one or more RNA sequences covalently linked to the template switching oligonucleotide and the UMI or unique sequence tag;(f) annealing a plurality of second RNA primers to the one or more RNA templates; and (g) extending the plurality of second RNA primers in a second nucleic acid extension reaction using a strand-displacement reverse transcriptase to generate a plurality of DNA sequence complementary to the one or more RNA templates, wherein the plurality of complementary DNA (cDNA) each comprise the complementary DNA sequence and a UMI or unique sequence tag. - View Dependent Claims (21, 22, 23, 24, 25, 27, 28, 29, 30)
-
Specification