Method and kit for the generation of DNA libraries for massively parallel sequencing

A parallel sequencing, large-scale technology, used in DNA preparation, recombinant DNA technology, biochemical equipment and methods, etc.

Pending Publication Date: 2019-03-15
MENARINI SILICON BIOSYSTEMS SPA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0019] -This method is not suitable for DNA derived from single cell samples

Method used

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  • Method and kit for the generation of DNA libraries for massively parallel sequencing
  • Method and kit for the generation of DNA libraries for massively parallel sequencing
  • Method and kit for the generation of DNA libraries for massively parallel sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0153] Scheme of LPWGS on Ion Torrent PGM after DRS-WGA

[0154] 1) Definitive restriction site whole genome amplification (DRS-WGA)

[0155] According to the manufacturer's instructions, use Ampli1 TM The WGA kit (Silicon Biosystems) amplifies single-cell DNA.

[0156] Ampli1 TM The WGA kit is designed to provide whole genome amplification from DNA obtained from a single cell. After cell lysis, the DNA is digested with a restriction enzyme, preferably MseI, and universal adapter sequences are ligated to the DNA fragments. Amplification is mediated by a single specific PCR primer for all generated fragments ranging in length from 200-1,000 bp distributed throughout the genome.

[0157] 2) Re-amplification of WGA products

[0158] 5 μL of WGA-amplified DNA was diluted by adding 5 μL of nuclease-free water and purified using the Agencourt AMPureXP system (Beckman Coulter) to remove unbound oligonucleotides and excess nucleotides, salts and enzymes.

[0159] Bead-based DNA...

Embodiment 2

[0200] Prototype for LPWGS on Ion Torrent Proton after DRS-WGA

[0201] 1. Definitive restriction site whole genome amplification (DRS-WGA)

[0202] Ampli1 was used according to the manufacturer's instructions as detailed in the previous example TM Single-cell DNA was amplified with the WGA kit (Menarini Silicon Biosystems).

[0203] 2. Double-Stranded DNA Synthesis

[0204] According to the manufacturing scheme, using Ampli1 TM The ReAmp / ds kit converts 5 μL of WGA-amplified DNA into double-stranded DNA (dsDNA). This process ensures the conversion of single-stranded DNA (ssDNA) molecules into dsDNA molecules.

[0205] 3. Purification of dsDNA products

[0206] 44 μL of nuclease-free water was added to dilute 6 μL of dsDNA synthesis product and purified by Agencourt AMPure XP beads (Beckman Coulter) to remove unbound oligonucleotides and excess nucleotides, salts and enzymes. Bead-based DNA purification was performed according to the following protocol: 75 μL (ratio: 1...

Embodiment 3

[0227] Protocol for Low-Pass Whole-Genome Sequencing on the Illumina MiSeq

[0228] plan 1

[0229] Definitive restriction site whole genome amplification (DRS-WGA):

[0230] According to the manufacturer's instructions, use Ampli1 TM The WGA kit (Silicon Biosystems) amplifies single-cell DNA. 5 μL of WGA-amplified DNA was diluted by adding 5 μL of nuclease-free water and purified using the Agencourt AMPure XP system (scale 1.8×). DNA was eluted in 12.5 μL and quantified by the dsDNA HS assay on a Qubit 2.0 fluorometer.

[0231] barcoded reamplification

[0232] Such as Figure 4 is schematically shown in, using Ampli1 TM Barcoding reamplification was performed with a PCR kit (Menarini Silicon Biosystems) in a volume of 50 μl. Each PCR reaction was composed as follows: 5 μl Ampli1 TM PCR reaction buffer (10×), 1 μL of one primer (25 μM) in SEQ ID NO: 195 to SEQ ID NO: 202, 1 μl of one primer (25 μM) in SEQ ID NO: 203 to SEQ ID NO: 214 primers, 1.75 μl Ampli1 TM ...

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Abstract

There is disclosed a method of generating a massively parallel sequencing library comprising the steps of :a) providing a primary WGA DNA library (pWGAlib), including fragments comprising a WGA library universal sequence adaptor; b)re-amplifying the primary WGA DNA library using at least one first primer (1PR) and at least one second primer (2PR); the at least one first primer (1PR) comprising from 5' to 3' at least one first sequencing adaptor (1PR5SA), at least one first sequencing barcode (1PR5BC) and a first primer 3' section (1PR3S) hybridizing to either the WGA library universal sequenceadaptor or its reverse complementary; the at least one second primer (2PR) comprising from 5' to 3' at least one second sequencing adaptor (2PR5SA) different from the at least one first sequencing adaptor (1PR5SA), and a second primer 3' section (2PR3S) hybridizing to either the WGA library universal sequence adaptor or its reverse complementary.

Description

technical field [0001] The present invention relates to methods and kits for generating massively parallel sequencing libraries for whole genome sequencing from whole genome amplification products (WGA). In particular, the method can also be applied to deterministic restriction sites (DeterministicRestriction-Site), whole genome amplification (DRS-WGA) DNA products. [0002] This library can be advantageously used for low-pass whole genome sequencing and genome-wide copy-number profiling. Background technique [0003] For single cells, it is useful to perform whole genome amplification (WGA) in order to obtain more DNA to simplify and / or enable different types of genetic analysis (including sequencing, SNP detection, etc.). [0004] WGA using definitive restriction site based LM-PCR (eg as described in WO / 2000 / 017390) is known in the art (hereinafter abbreviated as DRS-WGA). DRS-WGA has been shown to be a better protocol for the expansion of single cells (Reference: Lee YS...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12Q1/6869
CPCC12Q1/6806C12Q1/6855C12Q1/6869C12Q2525/155C12Q2525/191C12Q2535/122C12Q2563/143C12Q2563/149C12Q2563/179C12N15/1082
Inventor 尼科洛·马纳雷西热尼·布松保拉·托诺尼
Owner MENARINI SILICON BIOSYSTEMS SPA
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