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Method for analyzing heterozygosity deficiency (LoH) after genome-wide amplification of deterministic restriction site (DRS-WGA)

A technique of DRS-WGA, loss of heterozygosity, applied in the field of loss of heterozygosity in low-pass whole genome sequencing data analysis samples, which can solve the problem of insufficient information, inapplicability, and unreliable reanalysis of single cells And other issues

Pending Publication Date: 2022-05-10
MENARINI SILICON BIOSYSTEMS SPA
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0014] 2. In some applications, such as preimplantation genetic screening (PGS) or preimplantation genetic diagnosis (PGD), only single cells may be available, so the method of Zahn et al. is clearly not applicable
[0015] 3. In some applications, there may be multiple cells available for analysis, but they may still not be informative enough to use the method of Zahn et al.
[0021] -Inability to reliably reanalyze individual cells for validation or additional genomic information of interest

Method used

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  • Method for analyzing heterozygosity deficiency (LoH) after genome-wide amplification of deterministic restriction site (DRS-WGA)
  • Method for analyzing heterozygosity deficiency (LoH) after genome-wide amplification of deterministic restriction site (DRS-WGA)
  • Method for analyzing heterozygosity deficiency (LoH) after genome-wide amplification of deterministic restriction site (DRS-WGA)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0189] In Example 1, the Ampli1 LowPass for Illumina DNA library of 1 circulating tumor cell (CTC; test) and 1 white blood cell (WBC; control) obtained from a male patient affected by multiple myeloma was considered. Sequencing reads were mapped to the hg19 reference human genome and downsampled at 1, 2, 3, 4, 5, 6, 7, 8, 9 million reads. Alleles at the dbSNP polymorphic locus (common variants of dbSNP150 with a minor allele frequency ≥ 5%) were extracted from both libraries. Loci were segmented with a fixed 10,000,000 bp genomic window. One-sided Fisher's exact test was used to assess the significance of the association between the two categories (Table 4), with the null hypothesis that the heterozygous and homozygous loci were likely to be the same in WBC (control) and CTC (test).

[0190] Table 4

[0191]

[0192] The test results for each downsampling level are as follows Figure 15 shown. Starting with 2 million reads, the method demonstrated high sensitivity in de...

Embodiment 2

[0195] In Example 2, the same single CTC data used in Example 1 was used as input and the data was downsampled at 1 million reads. In this case, the loci were partitioned in the window into a fixed number (n = 1000) of loci covered by at least 1 read. For the identification of LoH regions, the LoH score was calculated as the number of heterozygous positions in each window.

[0196] Figure 16 Detection of LoH by using a genomic window with a constant number of loci is shown. In particular, the top panel shows the copy number map of the same CTCs as in Example 1. The x-axis is chromosomes; the y-axis is copy number. Each point represents a fixed-size window of the genome. Copy number bins are indicated by solid lines. Below the plot is a heatmap representing the heterozygous counts for each genomic window. Windows with lower LoH scores (lower number of heterozygous loci) are more likely to be in LoH status, indicated by darker shades of gray. Chromosome 11, the large arm...

Embodiment 3

[0200] In Example 3, two individual Hodgkin Reed / Sternberg (Hodgkin Reed / Sternberg, HRS) cells obtained from FFPE tissue of a classic Hodgkin lymphoma sample from a male patient were analyzed for their use in Ampli1 LowPass for Illumina Libraries. Two HRS cells share the same copy number profile. Sequencing reads were mapped to the hg19 reference human genome, and alleles present at the dbSNP polymorphic locus (common variants of dbSNP150 with a minor allele frequency ≥ 5%) were extracted from both libraries. Locus partitioning using copy number segments obtained by using Control-FREEC software, enabling GC-based normalization and segmentation of copy number signals [Boeva, V et al., Bioinformatics, 27(2), 268-269 .http: / / doi.org / 10.1093 / bioinformatics / btq635). An internal control defined jointly by all regions with a copy number equal to the ploidy of the cell (copy number=2) was used. For each segment defined by copy number analysis and contained in a chromosome arm, a on...

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Abstract

Disclosed is a method of analyzing a heterozygosity deletion (LoH) in at least one sample comprising genomic DNA, the method comprising the steps of: a. Providing said at least one sample comprising genomic DNA; b, carrying out deterministic restriction site whole genome amplification (DRS-WGA) on the genome DNA; c, preparing a large-scale parallel sequencing library from a product of the DRS-WGA; d, carrying out sequencing on the large-scale parallel sequencing library in the form of lt; performing low-pass whole genome sequencing according to the average coverage depth of 1; e. Aligning the reads obtained in step d on the reference genome of the at least one sample; f, extracting allele contents of a plurality of gene loci, wherein the plurality of gene loci comprise polymorphic gene loci and / or heterozygous gene loci; g. Assigning a LoH score to at least one genomic window of the reference genome for the at least one sample as a function of the number of loci having at least two different alleles among the plurality of loci.

Description

[0001] Cross References to Related Applications [0002] This patent application claims priority from Italian Patent Application No. 102019000013335 filed on July 30, 2019, the entire disclosure of which is incorporated herein by reference. technical field [0003] The present invention relates to a method for the analysis of loss of heterozygosity (LoH) in a sample from low-pass whole genome sequencing data from Definitive Restriction Site Whole Genome Amplification (DRS-WGA) with or without the use of normal controls , enabling single-cell resolution. The method can be applied to several single-cell applications, such as oncology, including the analysis of circulating tumor cells as well as single-cell heterogeneity in tissue samples; or reproductive medicine, including preimplantation genetic screening (PGS). Background technique [0004] Whole genome amplification (WGA) of single-cell genomic DNA is often required to obtain more DNA to simplify and / or allow different ty...

Claims

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Application Information

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IPC IPC(8): C12Q1/6869C12Q1/6886G16B20/20
CPCC12Q1/6869C12Q1/6886G16B20/20C12Q2525/191C12Q2521/301C12Q2535/122G16B30/10G16B20/10
Inventor 尼科洛·马纳雷西玛丽安娜·加龙齐阿尔贝托·费拉里尼克劳迪奥·福尔卡托
Owner MENARINI SILICON BIOSYSTEMS SPA
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