Methods and compositions for nucleic acid sample preparation
First Claim
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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.
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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.
16 Citations
27 Claims
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1. A method of performing a linear amplification of a plurality of RNA molecules, the method comprising:
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 23, 24, 25)
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13. A method of performing multiplex analysis of retroviral populations, the method comprising:
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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. - View Dependent Claims (14, 15, 16, 17, 18, 19, 20, 21, 22, 26, 27)
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Current AssigneePacific Bioscience of California Incorporated (L'Oreal SA)
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Original AssigneePacific Bioscience of California Incorporated (L'Oreal SA)
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InventorsVilfan, Igor, Turner, Stephen
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Primary Examiner(s)Chunduru, Suryaprabha
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Application NumberUS14/477,472Publication NumberTime in Patent Office1,244 DaysField of SearchNoneUS Class CurrentCPC Class CodesC12N 15/1096 cDNA Synthesis; Subtracted ...C12Q 1/6844 Nucleic acid amplification ...C12Q 1/6869 Methods for sequencingC12Q 2535/122 Massive parallel sequencing
