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Pre-anchor wash

An anchoring and washing technology, applied in the field of cleaning before anchoring, which can solve the problems of decreased mapping rate and increased inconsistency rate.

Inactive Publication Date: 2015-04-08
COMPLETE GENOMICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, after multiple cycles of DNA sequencing reactions are performed on DNA molecules arranged on a solid substrate, there is an increase in the discordance rate and a decrease in the mapping yield

Method used

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Examples

Experimental program
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preparation example Construction

[0119] Preparation of nucleic acid template of the present invention

[0120] Methods for preparing library constructs are described in detail, for example, in US Patent Application Publications 2010 / 0105052 and US2007099208, and US Patent Applications 11 / 679,124 (published in US2009 / 0264299); 11 / 981,761 (US2009 / 0155781); 11 / 981,661 (US2009 / 0005252); 11 / 981,605 (US2009 / 0011943); 11 / 981,793 (US2009-0118488); 11 / 451,691 (US2007 / 0099208); 11 / 981,607 (US2008 / 0234136); 11 / 982,467 (US2009 / 0137414); 11 / 451,692 (US2007 / 0072208); 11 / 541,225 (US2010 / 0081128; 11 / 927,356 (US2008 / 0318796); 11 / 938,106 (US2008 / 0171331); 10 / 547,214 (US2007 / 0037152); 11 / 981,730 (US2009 / 0005259); 11 / 981,685 (US2009 / 0036316); 11 / 981; / 934,695 (US2009 / 0075343); 11 / 934,697 (US2009 / 0111705); 11 / 934,703 (US2009 / 0111706); 12 / 265,593 (US2009 / 0203551); 0221832); 12 / 325,922 (US2009 / 0318304); 12 / 252,280 (US2009 / 0111115); 12 / 266,385 (US2009 / 0176652); 12 / 335,168 (US2009 / 0311691); 361,507 (US2009 / 0263802), 11 / 981,804 (...

Embodiment 1

[0368] Example 1: Making DNB

[0369] The following is an exemplary protocol for making DNBs (also referred to herein as "amplicons") from nucleic acid templates of the invention comprising a target nucleic acid with one or more interspersed adapters. First, a single-stranded linear nucleic acid template is amplified with a phosphorylated 5' primer and a biotinylated 3' primer, thereby forming a biotin-labeled double-stranded linear nucleic acid template.

[0370] First, by resuspending MagPrep-streptavidin magnetic beads (Novagen Part. No. 70716-3) in 1x magnetic bead binding buffer (150 mM NaCl and 20 mM Tris, pH=7.5 , in nuclease-free water) to prepare streptavidin magnetic beads. Place the tubes in a magnetic tube rack, allow the magnetic particles to clear, and remove and discard the supernatant. Then, wash the beads twice in 800 µl of 1x Bead Binding Buffer and resuspend in 80 µl of 1x Bead Binding Buffer. The amplified nucleic acid templates from the PCR reactions (a...

Embodiment 2

[0376] Example 2: Single c-PAL and double c-PAL

[0377] Fully degenerate second anchor probes of different lengths were tested in a two-anchor probe detection system. The combinations used were: (1) a standard one-anchor ligation using an anchor bound to the adapter adjacent to the target nucleic acid and a 9-mer unit sequencing probe, read at position 4 off the adapter; ( 2) read at position 9 away from the adapter using a two-anchor ligation comprising the same first and second anchors of the degenerate 5-mer and 9-mer sequencing probes; (3) using A two-anchor ligation comprising the same first and second anchors of the degenerate 6-mer and 9-mer sequencing probes, read at position 10 from the adapter; and (4) using Bi-anchor junctions of the same first and second anchors for the degenerate 8-mer and 9-mer sequencing probes, read at position 12 from the adapter. 1 μM of the first anchor probe and 6 μM of the degenerate second anchor probe were mixed with T4 DNA ligase i...

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Abstract

The present invention is directed to an aqueous wash solution comprising an acid and / or a cationic surfactant and its use in a method for improving the discordance rate and mapping yield in nucleic acid sequencing reactions.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Patent Application Serial No. 61 / 637,240, filed April 23, 2012, which is hereby incorporated by reference in its entirety for all purposes. Background technique [0003] Performing biochemical assays on nucleic acid molecules, such as DNA sequencing, for example, can subject the DNA molecules to harsh environments that affect the data obtained from such assays. For example, after multiple cycles of DNA sequencing reactions are performed on DNA molecules arranged on a solid substrate, there is an increase in the discordance rate and a decrease in the mapping yield. Contents of the invention [0004] The present invention relates to methods and compositions for improving discordance rates, mappability rates, and other metrics of nucleic acid sequencing reactions. Specifically, according to one embodiment of the present invention, a "pre-anchoring cleaning solution" is u...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6874C12Q2527/125
Inventor 马修·卡洛陈林苏丹尼斯·G·巴林格
Owner COMPLETE GENOMICS INC