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Identification of genomic structural variants using long read sequencing

A genome and variant technology, applied in genetic engineering, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of expensive whole genome sequencing and difficulty in analyzing structural variants

Pending Publication Date: 2022-05-27
BLACK HAWK GENOMICS LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Whole-genome sequencing using long-read sequencers can be used to detect large structural variants; however, whole-genome sequencing is expensive, and some long-read sequencers have difficulty resolving very large structural variants

Method used

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  • Identification of genomic structural variants using long read sequencing
  • Identification of genomic structural variants using long read sequencing
  • Identification of genomic structural variants using long read sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0404] Example 1: Target enrichment protocol

[0405] An exemplary target enrichment protocol begins with the preparation of the Cas9 ribonucleoprotein complex (RNP). All crRNAs were pooled into an equimolar mixture at a total concentration of 50-100 μM prior to guide RNA assembly. The crRNA mixture and tracrRNA were then combined so that both the tracrRNA concentration and the total crRNA concentration were 5-10 μM. The gRNA duplexes were formed by denaturation at 95°C followed by cooling to room temperature. Ribonucleoprotein complexes (RNPs) were constructed by combining gRNA duplexes with Cas9 nuclease followed by incubation at room temperature.

[0406] The next stage involves dephosphorylation of genomic DNA. From 1 to 4 genomic DNA samples can be combined into dephosphorylation reactions for a total of 1-5 μg gDNA per phosphorylation reaction. Input DNA was dephosphorylated using calf intestinal phosphatase or shrimp alkaline phosphatase.

[0407] The next stage in...

Embodiment 2

[0410] Example 2: BRCA1 crRNA probe design

[0411] In the case of BRCA1, the CHOPCHOP design program generated a total of 5567 possible crRNA probes along the entire length of the BRCA1 genomic locus. These crRNA sequences were then filtered using the filtering scheme described in [0041], reducing the number to 233 crRNA probes. The crRNA sequences are then checked using a second design checker tool such as the IDT CRISPR-Cas9 Guide RNA Design Checker Tool. The number of candidate crRNA probes was reduced to 86 probes. The final set of crRNA probes was selected based on the position of the target site.

[0412] like Figure 4A As shown, successful Cas9 cleavage and sequencing resulted in increased sequencing coverage of target regions, with little or no sequencing coverage of non-target regions. In samples with known deletions in exons 15 and 16, a sharp drop in sequencing coverage was observed where the deletions occurred ( Figure 4B ).

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Abstract

Provided herein are systems and methods for detecting genomic structural variants using non-applied gene editing sample preparation followed by long read sequencing.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS [0002] This application claims the benefit of US Provisional Application No. 62 / 913,886, filed October 11, 2019, and US Provisional Application No. 62 / 981,146, filed February 25, 2020, each of which is incorporated by reference Incorporated herein. Background technique [0003] Genetic abnormalities or genomic variations in an individual's genetic makeup can lead to an individual's genetic disease or disorder. Genetic abnormalities or genomic variations can range from discrete mutations in a single base (eg, single nucleotide variants) to chromosomal abnormalities or structural variants (SVs) (eg, copy number variants, segment inversions, etc.), including Rearrangement, addition or deletion of one or more genes. Currently, more than 100,000 genetic variants have been classified as pathogenic in public databases. For example, sickle cell disease is caused by single-nucleotide mutations in the beta-globin gene; Fragile X syndr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6811C12Q1/6806C12Q1/6869
CPCC12Q1/6869C12Q1/6811C12Q2521/301C12Q2565/631C12Q2525/191C12Q1/6827C12N2310/20C12Q2537/159C12Q2561/113C12Q2563/143C12Q2563/149C12Q2535/122C12Q1/6806C12Q1/6858C12Q1/6883C12Q2600/156
Inventor 奥瓦萨·塔图姆李知恩
Owner BLACK HAWK GENOMICS LLC
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