Methods and compositions for enrichment of target nucleic acids
Inventors
Wang, Chunlin • MA, Zhihai • Gharizadeh, Baback
Assignees
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Publication Number
US-11261479-B2
Publication Date
2022-03-01
Expiration Date
Abstract
The present disclosure relates to a method of target enrichment and amplification of selective target regions of genomes or nucleic acid samples and further analysis by systems such as NGS. The disclosure provides methods and kits that can be used in numerous downstream procedures including DNA sequencing. The disclosed methods can be utilized to sequence the target nucleic acid sequences such as to detect the presence of genetic variations for biological assays, assessment of disease, to count copies of target regions, and to allow such target enrichment prior to sequencing.
Core Innovation
The invention provides a method for enrichment of target nucleic acid sequences that uses directionally ligating partially double-stranded blocking adapters to the ends of one or more nucleic acid samples. The method uses a biotin-labeled common primer A together with forward strand-specific primers that comprise universal sequence B in a first test reaction, and a second portion of the ligated nucleic acid samples is combined with the biotin-labeled common primer A and one or more reverse strand-specific primers that comprise universal sequence B in a second test reaction.
Each test reaction is subjected to amplification to generate a first set of amplicons. The first set of amplicons is enriched by pooling the first set of amplicons and binding the pooled first set of amplicons to streptavidin-coated beads. A portion of the pooled first set of amplicons is then subjected to a second round of amplification using common primer A and common primer B to generate a final set of amplicons.
In embodiments described in the document, the partially double-stranded blocking adapters include strand L and strand S features such as barcode and unique molecular identifier, and the workflow is extended with optional processing including end repair and dA-tailing and RNA reverse transcription to ds cDNA with fragmentation. The document also describes downstream next-generation sequencing with software allele-count analysis at polymorphic sites and design principles intended to minimize primer-dimers and off-target amplification while reducing target dropout using primer compatibility scoring.
Claims Coverage
The document contains two independent claims: a two-round enrichment method and a corresponding kit. The inventive features are directed to directionally ligating partially double-stranded blocking adapters, performing separate forward- and reverse-strand-specific primer amplifications using universal sequence B, biotin/streptavidin capture, and a final amplification with common primer A and common primer B; and, in the kit claim, to providing complementary adapter and primer components with the specified universal/barcode relationships.
Directionally ligating partially double-stranded blocking adapters to sample ends
directionally ligating partially double-stranded blocking adapters to the ends of one or more nucleic acid samples
Separate forward and reverse strand-specific primer amplification using universal sequence B
combining a portion of the ligated nucleic acid samples with a biotin-labeled common primer A and one or more forward strand-specific primers in one test reaction, and combining a second portion with the biotin-labeled common primer A and one or more reverse strand-specific primers in a second test reaction, wherein the forward and reverse strand-specific primers comprise universal sequence B
Pooling, biotin/streptavidin enrichment, and second-round common primer amplification
subjecting each test reaction to amplification to generate a first set of amplicons; enriching the first set of amplicons by pooling the first set of amplicons together and then binding the pooled first set of amplicons to streptavidin-coated beads; and subjecting a portion of the first set of amplicons to a second round of amplification using common primer A and common primer B to generate a final set of amplicons
Kit with partially double-stranded blocking adapters carrying barcode and universal sequence A plus universal-sequence-B strand-specific primers
a kit comprising at least one partially double-stranded blocking adapter wherein strand L comprises a barcode and a universal sequence A, at least one forward strand-specific primer, at least one reverse strand-specific primer, a biotin-labeled common primer A, and common primer A and common primer B, wherein the forward and reverse strand-specific primers each comprise universal sequence B
Overall claim coverage centers on a two-round enrichment and amplification workflow that combines strand-specific primers (universal sequence B) in separate forward and reverse test reactions with biotin-labeled common primer A, streptavidin capture of first-round amplicons, and a final second-round amplification with common primer A and common primer B; and on a kit that provides the required adapter and primer components, including barcode/universal sequence A on strand L and universal sequence B on both forward- and reverse-strand-specific primers.
Stated Advantages
Enrichment of target nucleic acid sequences with a workflow that includes pooling and binding to streptavidin-coated beads.
Reduces primer-dimers and off-target amplification and reduces target dropout using primer compatibility scoring (as described in the document summary).
Documented Applications
Targeting disease/cancer-associated mutations and analyzing allele counts at polymorphic sites using downstream next-generation sequencing with software.
Copy number variation (CNVs) and fusion detection (including examples described as fusion genes).
Use with cfDNA/ctDNA, including prenatal/cell-free fetal DNA (maternal and fetal DNA).
Inherited disease/newborn screening and forensics (as described in the document summary).
Cancer gene panel testing (example described as a cancer gene panel with 327 primers using two parallel multiplex PCRs with forward and reverse strand reactions and biotin-streptavidin enrichment).
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