Sample preparation methods, systems and compositions
Inventors
Blauwkamp, Timothy A. • Sit, Rene • Vilfan, Igor D.
Assignees
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Publication Number
US-10697008-B2
Publication Date
2020-06-30
Expiration Date
Abstract
The disclosure provides methods, compositions, systems, and kits for the concurrent detection and analysis of different structural and chemical forms of nucleic acids in a sample.
Core Innovation
The invention relates to nucleic-acid sample preparation and sequencing workflows that enable concurrent analysis of multiple nucleic-acid structural and chemical forms, including DNA and RNA, having different single-stranded and double-stranded forms. It addresses analyzing a sample that comprises a mixture of single-stranded DNA and single-stranded RNA without physically splitting the sample into separate DNA and RNA workflows.
A first adapter is attached to the single-stranded DNA and a second adapter is attached to the single-stranded RNA. A first primer is annealed to the first adapter and a second primer is annealed to the second adapter, followed by extending the annealed first primer on the single-stranded DNA to form double-stranded DNA.
The workflow further extends the annealed second primer on the single-stranded RNA to form a double-stranded DNA-RNA hybrid. In described embodiments, non-templated nucleotides or overhangs create distinct tag types for DNA versus RNA, directing subsequent adapter hybridization or ligation and enabling identification of origin after sequencing, including through sequencing adapters and barcode or index identifying sequences.
The approach includes concurrent versus successive enzyme addition modes and adapter and enzymatic designs using specific ligases and polymerase or reverse transcriptase sets, with detection via sequencing, qPCR, dPCR, and microarrays.
Claims Coverage
The relevant independent claim is clm-00001, which recites a primer extension reaction on a sample containing both single-stranded DNA and single-stranded RNA by adapter attachment, annealing of respective primers, and distinct extensions that form double-stranded DNA and a DNA-RNA hybrid. Additional inventive features are described in dependent claims as refinements to adapter ligation and to extension conditions using specified enzyme types and non-templated nucleotide overhang features.
Primer extension on mixed single-stranded DNA and single-stranded RNA to form dsDNA and DNA-RNA hybrid
A method providing a sample comprising a mixture of single-stranded DNA and single-stranded RNA, attaching a first adapter to the single-stranded DNA and attaching a second adapter to the single-stranded RNA, annealing a first primer to the first adapter and annealing a second primer to the second adapter, extending the annealed first primer on the single-stranded DNA to form double-stranded DNA, and extending the annealed second primer on the single-stranded RNA to form a double-stranded DNA-RNA hybrid.
Adapter ligation using selected ligases
Ligation of a first adapter using a ligase selected from CircLigase II, Thermostable App-DNA/RNA ligase, T4 RNA ligase 1, T4 RNA Ligase 2 truncated, or any combination thereof.
DNA primer extension using Bst 2.0 DNA polymerase
Extending the annealed first primer on single-stranded DNA using Bst 2.0 DNA polymerase.
RNA primer extension using reverse transcriptase
Extending the annealed second primer on single-stranded RNA using reverse transcriptase.
Non-templated nucleotide addition at a 3' end
Adding at least one non-templated nucleotide to the 3' end.
Non-templated nucleotide length constraint
The at least one non-templated nucleotide has a length of up to eight nucleotides.
Across the independent and dependent claim set provided, the inventive coverage centers on performing primer extension on a mixed single-stranded DNA and single-stranded RNA sample via adapter attachment and parallel extensions that generate both double-stranded DNA and a double-stranded DNA-RNA hybrid, with further refinements specifying adapter ligation ligases, extension enzyme types, and optional non-templated 3' overhang features with a defined length limit.
Stated Advantages
Efficient recovery and reduced sample/input requirements.
Documented Applications
Pathogen nucleic acids.
Cancer diagnosis.
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