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System and methods for detecting genetic variation

A genetic variation and sequencing technology, applied in the field of systems and methods for detecting genetic variation, can solve problems such as low efficiency, wasting samples, reducing reliability and repeatability

Inactive Publication Date: 2018-08-28
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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  • System and methods for detecting genetic variation
  • System and methods for detecting genetic variation
  • System and methods for detecting genetic variation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0288] Example 1: Sample Preparation and Sequencing Methods

[0289] Genomic DNA (gDNA) was extracted in a 96-well format, leaving wells A1, G12, and H12 empty (which will later contain a no-template control, respectively, a general Negative standards, and samples containing one of several known causal genetic variants). Transfer 50 μL from each well to the corresponding well of the absorber plate. The absorbance at 260 nm was measured using a Tecan M200 plate reader to calculate the amount of DNA. Transfer 50 μL of gDNA from the absorber plate to an Eppendorf twin.tec plate. Control samples were added to their respective positions on the twin.tec plate. gDNA and controls were fragmented in a SonicMan (Matrical, Spokane WA) sonicator at 10°C according to the following protocol: precool 180 s, cycle 100, sonicate 3.0 s, power 35%, cover cool 1.0 s, plate cool 0 s, Post cooldown of 0 seconds. 2 μL samples were analyzed for fragment size distribution using a Fragment Analyze...

Embodiment 2

[0296] Example 2: Amplification and sequencing methods

[0297] Exemplary methods for amplifying multiple different target polynucleotides are shown in figure 2 with 5 , the main difference is that figure 2 Including a solid phase purification step. Figure 7 Exemplary amplification methods are also illustrated, and figure 2 The method exemplified in differs primarily in that oligonucleotide primer extension is performed prior to adapter ligation rather than after adapter ligation. Amplification may or may not include a solid phase purification step. Figure 6 exemplifies Figure 5 Amplification methods shown in , and also exemplary bridge amplification and sequencing methods. Can be Figure 6 The amplification methods exemplified in are used in conjunction with any bridge amplification and related sequencing methods.

[0298] First, partial single-stranded adapters are ligated to fragmented polynucleotides. Partial single-stranded adapters have at one end a double...

Embodiment 3

[0300] Example 3: Identification of non-subject sequences

[0301] Polynucleotides (eg, DNA and / or RNA) are extracted from a sample from a subject suspected of containing viral and / or bacterial polynucleotides using standard methods known in the art. The sample polynucleotides are fragmented, end modified and tailed (eg in Example 1). An adapter oligonucleotide comprising sequence D is then joined to the sample polynucleotide, which is then amplified using amplification primers comprising sequence C, sequence D and a barcode. The amplified target polynucleotide is hybridized to a plurality of different first oligonucleotides attached to the solid surface. Each first oligonucleotide comprises 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 comprises The sequence of the non-subject sequence or a sequence within 200 nucleotides of the non-subject sequence is compleme...

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Abstract

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

Description

[0001] This application is a divisional application of a Chinese patent application with an application date of July 17, 2013, an application number of 201380045879.5, and an invention title of "System and Method for Detecting Genetic Variation". Background of the invention [0002] Next-generation sequencing (NGS) allows small, inexpensive genome sequencing with turnaround times measured in days. However, as is generally performed and understood with NGS, all regions of the genome are sequenced with roughly equal probability, meaning that a large number of genome sequences are collected and discarded to collect sequence information from a relatively low percentage of regions, in The function in this region is well understood to allow interpretation of potential mutations. In general, only those regions of interest are purified from whole-genome samples as a separate step from sequencing. At the current state of the art it is usually an inefficient method that lasts for days....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6827C12Q1/6869C12Q1/6874
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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