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Systems and methods for detecting genetic variation

A technology for genetic variation and sequencing, applied in the field of systems and methods for detecting genetic variation, which can solve the problems of wasted samples, low efficiency, reduced reliability and reproducibility, etc.

Inactive Publication Date: 2018-04-27
COUNSYL INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, modifications to the sequencing substrate and accompanying library preparation according to previous recommendations lead to inefficiencies, reduced reliability and reproducibility, and waste of precious samples

Method used

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  • Systems and methods for detecting genetic variation
  • Systems and methods for detecting genetic variation
  • Systems and methods for detecting genetic variation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0169] Example 1: Sample preparation and sequencing method

[0170] Genomic DNA (gDNA) is extracted in 96-well format, leaving wells A1, G12, and H12 vacant (which will later contain no-template controls, respectively, containing Coriell samples lacking each pathogenic genetic variant tested. NA12878 genomic DNA universal Negative standards, and samples containing one of multiple known pathogenic genetic variants). Transfer 50 μL from each well to the corresponding well of the absorption plate. A Tecan M200 plate reader was used to measure the absorbance at 260nm to calculate the amount of DNA. Transfer 50 μL of gDNA from the absorption plate to the Eppendorf twin.tec plate. Add control samples to their respective positions on the twin.tec board. Fragment gDNA and control in a SonicMan (Matrical, Spokane WA) sonicator at 10°C according to the following protocol: pre-cooling for 180 seconds, cycling 100 times, ultrasound for 3.0 seconds, power 35%, lid cooling for 1.0 second, p...

Embodiment 2

[0177] Example 2: Amplification and sequencing method

[0178] An exemplary method for the amplification of multiple different target polynucleotides is shown in figure 2 with 5 , The main difference is figure 2 It includes a solid phase purification step. Figure 7 An exemplary amplification method is also illustrated, and figure 2 The main difference in the methods exemplified in is that oligonucleotide primer extension is performed before adaptor coupling rather than after adaptor coupling. Amplification may or may not include a solid phase purification step. Image 6 Exemplified Figure 5 The amplification methods shown in, as well as exemplary bridge amplification and sequencing methods. Can be Image 6 The amplification method exemplified in is used in combination with any bridge amplification method and related sequencing methods.

[0179] First, part of the single-stranded adaptor is ligated to the fragmented polynucleotide. Part of the single-stranded adaptor has a dou...

Embodiment 3

[0181] Example 3: Identification of non-subject sequences

[0182] Using standard methods known in the art, polynucleotides (e.g., DNA and / or RNA) are extracted from samples from subjects suspected of containing virus and / or bacterial polynucleotides. The sample polynucleotide is fragmented, end modified and tailed (for example in Example 1). The adaptor oligonucleotide containing sequence D is then ligated to the sample polynucleotide, and then amplified using amplification primers containing sequence C, sequence D and barcode. The amplified target polynucleotide is hybridized with a plurality of different first oligonucleotides attached to the solid surface. Each first oligonucleotide contains sequence A and sequence B, wherein sequence B is different for each different first oligonucleotide, is located at the 3'end of each first oligonucleotide, and contains The sequence of the non-subject sequence or the sequence within 200 nucleotides of the non-subject sequence is complem...

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Abstract

The present invention provides methods, devices and compositions for high-throughput amplification sequencing of specific target sequences in one or more samples. In some aspects, the barcode-tagged polynucleotides are sequenced simultaneously and the source of the sample is identified based on the barcode sequence. In some aspects, sequencing data is used to determine one or more genotypes of one or more loci comprising a causal genetic variant. In some aspects, systems and methods for detecting genetic variation are provided.

Description

Background of the invention [0001] Next-generation sequencing (NGS) allows small, inexpensive genome sequencing, and its turnaround time is calculated in days. However, just like the general implementation and understanding of NGS, all regions of the genome are sequenced with roughly equal probability, which means that a large number of genome sequences are collected and discarded to collect sequence information from a relatively low percentage of regions. The functions in these regions have been fully understood to interpret potential mutations. Generally speaking, as a separate step from sequencing, only those regions of interest are purified from whole genome samples. It is usually an inefficient method that lasts several days at the current state of the art. [0002] Direct targeted sequencing (DTS) is a modification of a standard sequencing scheme adopted by Illumina, which also allows the sequencing substrate (ie, flow cell) to become a genomic sequence capture substrate. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C40B40/06
CPCC12Q1/6827C12Q1/6869C12Q1/6874C12Q2525/155C12Q2525/161C12Q2537/143C12Q2563/179C12Q2565/543C12Q2535/122C12Q2565/514
Inventor H.理查兹E.埃文斯巴拉吉.斯里尼瓦桑萨布拉曼亚姆.斯里尼瓦桑A.沙A.S.帕特森C.查
Owner COUNSYL INC
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