Methods and compositions for nucleic acid sample preparation

Patent metadata

Field	Value
Publication number	US-9879318-B2
Application number	US-201414477472-A
Country	US
Kind code	B2
Filing date	Sep 4, 2014
Priority date	Sep 6, 2013
Publication date	Jan 30, 2018
Grant date	Jan 30, 2018

Abstract

The present invention provides methods and compositions useful for supplying high throughput nucleic acid sequencing systems with templates. The methods circumvent the need for costly, labor-intensive cloning and cell culture methods and can be scaled to accommodate template production for a variety of sequencing applications, e.g., sequencing individuals' genomes, sequencing subpopulations of transcripts from a gene of interest, and/or gene expression profiling. Particularly preferred embodiments of the methods vastly improve the preparation of cDNA from mRNA samples, e.g., by randomizing errors introduced during the process, thereby allowing these errors to be readily distinguished from true variants present in the mRNA samples.

First claim

What is claimed is:

1. A method of performing a linear amplification of a plurality of RNA molecules, the method comprising:
a) providing a sample comprising a plurality of RNA molecules, wherein the plurality of RNA molecules comprises RNA molecules having differing nucleotide compositions and wherein each of the plurality of RNA molecules comprises a 3′ end;
b) linking an adaptor to the 3′ end of each of said plurality of RNA molecules, wherein each adaptor comprises a barcode sequence and a Phi6 RNA replicase initiation sequence, wherein the barcode sequence is positioned 5′ to the Phi6 RNA replicase initiation sequence, and further wherein each adaptor has a different barcode sequence;
c) synthesizing a complementary nascent RNA strand for each of the plurality of adaptor-linked RNA molecules by contacting the plurality of adaptor-linked RNA molecules with a Phi6 RNA replicase, thereby generating double-stranded RNA molecules;
d) providing an oligonucleotide complementary to a segment of the first nascent RNA strand of each of the double-stranded RNA molecules, wherein the segment is complementary to at least a portion of the adaptor;
e) annealing the oligonucleotide to the first nascent RNA strand of each of the double-stranded RNA molecules, thereby separating the 5′ end of each of the first nascent RNA strands from the 3′ end of each of the plurality of adaptor-linked RNA molecules;
f) repeating said synthesizing, whereby the first nascent RNA strand is displaced and a second nascent RNA strand is synthesized; and
g) repeating said annealing and said synthesizing multiple times, thereby performing linear amplification of the plurality of adaptor-linked RNA molecules and producing a pool of amplified RNA molecules.

2. The method of claim 1 , wherein said multiple times is at least ten times.

3. The method of claim 1 , wherein the oligonucleotide is an LNA oligonucleotide.

4. The method of claim 1 , wherein the oligonucleotide is 5′-adenylated.

5. The method of claim 1 , wherein the adaptor is dideoxy-modified on the 3′ end.

6. The method of claim 1 , wherein the barcode sequence comprises randomized bases.

7. The method of claim 1 , further comprising converting the pool of amplified RNA molecules to cDNA.

8. The method of claim 1 , further comprising determining nucleotide sequences for the pool of amplified RNA molecules, wherein the nucleotide sequences comprise sequences that descended from the plurality of RNA molecules and barcode sequences.

9. The method of claim 8 , further comprising using the barcode sequences to link each of the nucleotide sequences to a single parental adaptor-linked RNA molecule.

10. The method of claim 1 , wherein the annealing is carried out at 40° C. and the synthesizing is carried out at 32° C.

11. The method of claim 1 , wherein the annealing and synthesizing are carried out at a single temperature that is at least five degrees higher than an optimal temperature for the Phi6 RNA replicase.

12. The method of claim 11 , wherein the temperature is 39-41° C.

13. A method of performing multiplex analysis of retroviral populations, the method comprising:
a) providing a sample comprising a plurality of linear RNAs from a retroviral population, wherein the retroviral population comprises multiple viral genomes having a different set of sequence variants, and wherein each linear RNA comprises a 3′ end;
b) linking an adaptor to the 3′ end of each of said linear RNA, wherein the adaptor comprises a barcode sequence and a Phi6 RNA replicase initiation sequence, wherein the barcode sequence is positioned 5′ to the Phi6 RNA replicase initiation sequence, and further wherein each adaptor has a different barcode sequence, thereby generating adaptor-linked viral RNAs;
c) synthesizing first nascent RNA strands for each of the adaptor-linked viral RNAs by contacting said adaptor-linked viral RNAs with a Phi6 RNA replicase, thereby generating double-stranded RNA molecules;
d) providing an oligonucleotide complementary to a segment of the first nascent RNA strands of each of the double-stranded RNA molecules, wherein the segments are complementary to at least a portion of the adaptor;
e) annealing the oligonucleotide to the first nascent RNA strands of each of the double-stranded RNA molecules, thereby separating the 5′ ends of the first nascent RNA strands from the 3′ ends of the adaptor-linked viral RNAs;
f) repeating said synthesizing, whereby the first nascent RNA strands are displaced and second nascent RNA strands are synthesized;
g) repeating said annealing and said synthesizing multiple times, thereby performing linear amplification of the adaptor-linked viral RNAs and producing multiple nascent RNA strands complementary to each of the adaptor-linked viral RNAs;
h) converting the multiple nascent RNA strands complementary to the adaptor-linked viral RNAs into cDNAs, thereby generating a pool of cDNAs in which all members of the pool of cDNAs that are descended from the same adaptor-linked viral RNA comprise identical barcode regions;
i) determining nucleotide sequences for the members of the pool of cDNAs, wherein the nucleotide sequences comprise adaptor-linked viral RNA sequences and barcode sequences;
j) grouping the nucleotide sequences based on the barcode sequences, wherein all nucleotide sequences from members of the pool of cDNAs that are descended from the same adaptor-linked viral RNA are grouped together, thereby composing one group of nucleotide sequences for each of the adaptor-linked viral RNAs; and
k) using the adaptor-linked viral RNA sequences in each group composed in j) to construct a consensus sequence for each of the adaptor-linked viral RNAs.

14. The method of claim 13 , wherein the plurality of linear RNAs comprises fragmented viral genomes.

15. The method of claim 13 , wherein the plurality of linear RNAs comprises full-length viral genomes.

16. The method of claim 13 , further comprising amplifying the pool of cDNAs prior to determining their nucleotide sequences.

17. The method of claim 13 , wherein the synthesizing the nascent RNA strands is imperfect such that some of the nascent RNA strands comprise errors, and further wherein the consensus sequences do not comprise the errors in the nascent RNA strands, but do comprise the sequence variants present in the adaptor-linked viral RNAs from which the cDNAs descended.

18. The method of claim 13 , wherein the converting the multiple nascent RNA strands complementary to the adaptor-linked viral RNAs into cDNAs is imperfect such that some of the cDNAs comprise errors, and further wherein the consensus sequences do not comprise the errors in the cDNAs, but do comprise the sequence variants present in the adaptor-linked viral RNAs from which the cDNAs descended.

19. The method of claim 13 , wherein the oligonucleotides are LNA oligonucleotides.

20. The method of claim 13 , wherein the oligonucleotides are 5′-adenylated.

21. The method of claim 13 , wherein the adaptor is dideoxy-modified on the 3′ end.

22. The method of claim 13 , wherein the barcode sequence comprises randomized bases.

23. The method of claim 1 , wherein the Phi6 RNA replicase initiation sequence is 5′-UUUUUUUCC-3′.

24. The method of claim 23 , wherein the oligonucleotide comprises a 3′ sequence selected from: a TTTTTTTCC-3′ DNA sequence and a UUUUUUUCC-3′ RNA sequence.

25. The method of claim 9 , further comprising grouping the nucleotide

Assignees

• Pacific Biosciences California Inc

Inventors

• Vilfan Igor
• Turner Stephen

Classifications

• C12Q2535/122

  Massive parallel sequencing · CPC title

• C12N15/1096

  cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title