Compositions and methods for library construction and sequence analysis

A library and sequence technology, applied in the field of compositions and methods for library construction and sequence analysis, can solve problems such as loss of sensitivity, low efficiency, and limited accuracy

Pending Publication Date: 2020-02-07
SINGLERA GENOMICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This limits the accuracy when calling low allele ratio mutants, as the low efficie...

Method used

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  • Compositions and methods for library construction and sequence analysis
  • Compositions and methods for library construction and sequence analysis
  • Compositions and methods for library construction and sequence analysis

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0202] In this embodiment, the templates (eg, polynucleotides to be sequenced) are short DNA fragments less than about 200 bp long. These DNA fragments may include DNA extracted from plasma, enzymatically treated (eg, by fragmentation enzymes) genomic DNA, or physically sheared DNA. Physically sheared DNA can undergo end repair. In certain aspects, the template has a 3' hydroxyl for attachment.

[0203] Typically, for example, using 1 U FastAP thermosensitive alkaline phosphatase (Thermo Scientific) in 100 mM MOPS (pH 7.5), 20 mM KCl, 10 mM MgCl 2 , 2mM DTT and 5mM MnCl 2 Phosphorylate 10-30 ng of properly prepared template DNA for 10 min at 37°C. The DNA was then denatured at 95°C for 2 min and then placed on ice for 1 min.

[0204] A single-chain linker with a 5' phosphate group and a 3' carbon spacer was synthesized by IDT. The 5' end contains GA followed by the 12-mer's Unique Molecular Identifier (UMI) sequence. A typical single-chain linker has the following sequen...

Embodiment 2

[0215] In this example, genomic DNA (gDNA) samples with known variants and plasma samples were tested using 10 ng and 20 ng input amounts. gDNA samples contain single nucleotide variants (SNVs, which can be used interchangeably with "single nucleotide change" SNCs), insertion deletions (indels), CNVs, and fusions. Each variant was called at different allelic fractions: 5%, 1%, 0.5% and 0.1%. Sensitivity and specificity were measured for each mutation type at each allele fraction. The primer pool used here is shown in Table 1. Each target-specific primer can be used in the same volume ratio or in different volume ratios throughout the pool. For example, for primers with a volume ratio of 2, the primers should be added at twice the volume of the primers with a ratio of 1.

[0216] Table 1:

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[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231] In one aspe...

Embodiment 3

[0242] In this example, a method is described for the construction of libraries from extracted plasma DNA, for example to interrogate single nucleotide changes (SNCs), insertion deletions (indels), copy number variations (CNVs) from circulating tumor DNA. ) and fusion. As a rationale for this example, extracted plasma DNA (eg, from a human) is dephosphorylated and denatured. Single-stranded DNA ligation adds a universal adapter to the 3' end of each molecule. The DNA is then subjected to semi-targeted PCR using site-specific primers for the adapters and a reverse complementary primer. Libraries were prepared using secondary PCR to add full-length adapters and barcodes to each molecule.

[0243] Equipment, materials, and consumables used in this example include: Veriti thermal cycler, 96-well magnet, 96-well cryoblock, vortexer, well-plate microcentrifuge, semi-skirted 96-well PCR plate, plate seals, pipette Pipettes and pipette tips.

[0244] The reagents and media used in...

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Abstract

The present disclosure relates to methods for constructing polynucleotide libraries and/or polynucleotide sequencing. Related kits and devices are also disclosed. The present disclosure also relates to compositions, kits, devices, and methods for conducting genetic and genomic analysis, for example, by polynucleotide sequencing. In particular aspects, provided herein are compositions, kits, and methods for constructing libraries with improved ligation efficiency and conversion rate during sequencing. In certain embodiments, the compositions, kits, and methods herein are useful for analyzing polynucleotide fragments, such as circulating polynucleotide fragments in the body of a subject, Including circulating tumor DNA

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of priority to U.S. Provisional Application Serial No. 62 / 487,423, filed April 19, 2017, and U.S. Provisional Application Serial No. 62 / 657,544, filed April 4, 2018, the contents of which are incorporated by reference incorporated in its entirety and for all purposes. In certain aspects, this disclosure is related to US Provisional Application Serial No. 62 / 487,422, filed April 19, 2017, the contents of which are incorporated by reference in their entirety for all purposes. technical field [0003] The present disclosure relates to compositions, kits, devices and methods for gene and genome analysis, eg, by polynucleotide sequencing. In particular aspects, provided herein are compositions, kits, and methods for constructing libraries with improved ligation efficiency and conversion rates during sequencing. In certain embodiments, the compositions, kits and methods herein can be used ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12Q1/6844C12Q1/6853C12Q1/6855C40B50/14C12N15/10
CPCC12Q1/6806C12N15/1093C40B40/06C12Q2521/501C12Q2525/191C12Q2535/122C12Q2563/179C40B50/14C12Q1/6874
Inventor 杰弗里·A·戈尔阿瑟瓦·高赫刘蕊
Owner SINGLERA GENOMICS INC
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