Single-Cell DNA Sequencing for Mixture Deconvolution
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Questions and Answers

What was the concentration of DNA used as input for SNP microarray analysis?

50 ng/µL

Which platform was used to process the SNP array data?

Genome Studio 2.0

How many high-quality DNA samples were used to create a pre-existing cluster file?

4405

What was the total number of SNPs left for analysis after removing variants with missing genotypes?

<p>719,923</p> Signup and view all the answers

What reference genome was used to set the reference alleles in PLINK v2.00?

<p>GRCh38</p> Signup and view all the answers

Which tool was used to overlap the identity SNP lists obtained from scATAC-seq data with the WES and GSA reference data?

<p>bcftools isec</p> Signup and view all the answers

Why was individual genetic identification not possible from in-silico mixture?

<p>Lack of reference data</p> Signup and view all the answers

How were the correct individual-specific mixture deconvolutions assessed?

<p>Based on the barcodes known to belong to one individual or the other</p> Signup and view all the answers

What specific technique was used in this study to perform non-targeted single-cell DNA sequencing (scDNA-seq)?

<p>The study employed scATAC-seq (Assay for Transposase-Accessible Chromatin with sequencing) for non-targeted single-cell DNA sequencing.</p> Signup and view all the answers

What is the significance of separating cells from biological mixtures in forensic science?

<p>Separating cells from biological mixtures allows for the individual identification of contributors to the mixture, which is crucial for solving crimes and establishing paternity.</p> Signup and view all the answers

What was the complexity of the blood mixtures tested in this study?

<p>The study tested mixtures of five individuals, encompassing both sexes and diverse biogeographic ancestries.</p> Signup and view all the answers

What are the two main methods of single-cell DNA analysis mentioned in the text?

<p>The two main methods of single-cell DNA analysis mentioned are scRNA-seq (single-cell RNA sequencing) and scDNA-seq (single-cell DNA sequencing).</p> Signup and view all the answers

What key information was successfully extracted from the deconvoluted mixture samples?

<p>The study successfully determined the genetic sex and biogeographic ancestry of each individual within the mixtures, establishing their individual identities.</p> Signup and view all the answers

Explain the advantage of scDNA-seq over scRNA-seq in terms of SNP detection.

<p>scDNA-seq detects more SNPs from fewer cells compared to scRNA-seq, providing higher sensitivity for forensic applications.</p> Signup and view all the answers

Describe the in-silico experiments conducted to further test the efficacy of the approach.

<p>The in-silico experiments evaluated the approach's ability to deconvolute highly complex mixtures with up to 11 individuals, balanced mixtures with as few as 20 cells (10 per individual), and imbalanced mixtures with ratios as low as 1:80.</p> Signup and view all the answers

What is the potential application of compound markers (two or more autosomal SNPs) in forensic genetics?

<p>Compound markers can be used for forensic mixture deconvolution, especially when analyzed with targeted MPS (Massively Parallel Sequencing).</p> Signup and view all the answers

What is the significance of the in-silico results regarding mixture complexity?

<p>The in-silico results indicate that the scATAC-seq approach is capable of resolving highly complex mixtures, mixtures with low cell counts, and imbalanced mixtures, suggesting its broad applicability in forensic settings.</p> Signup and view all the answers

What is a major limitation of using compound markers for forensic purposes?

<p>The availability of reference samples from the suspects is required to generate reference data for analysis.</p> Signup and view all the answers

What data analysis pipeline was used in this study to deconvolute the single-cell DNA sequencing data?

<p>The study utilized the De-goulash data analysis pipeline to deconvolute the single-cell DNA sequencing data.</p> Signup and view all the answers

What is the overall conclusion of this study regarding the use of scDNA-seq, specifically scATAC-seq, in forensic applications?

<p>The study concludes that scDNA-seq, particularly scATAC-seq, is a viable method for mixture deconvolution, genetic characterization, and individual identification in forensic investigations.</p> Signup and view all the answers

What is the significance of the date "Received 22 August 2023" in the context of the research article?

<p>It indicates the date when the research article was first submitted for publication to the journal.</p> Signup and view all the answers

Explain the potential impact of this study on forensic science.

<p>This study suggests that single-cell DNA sequencing methods like scATAC-seq could revolutionize forensic investigations by enabling the analysis of complex mixtures, leading to more precise individual identification and solving cold cases.</p> Signup and view all the answers

What is the meaning of the CC BY license mentioned in the article?

<p>The CC BY license means the article is an open access resource, allowing others to share, adapt, and build upon the work, as long as they credit the original authors.</p> Signup and view all the answers

Identify the two departments at Erasmus MC University Medical Center Rotterdam that are associated with the authors of the research article.

<p>The departments are Department of Genetic Identification and Department of Cell Biology.</p> Signup and view all the answers

How many single-individual scATAC-seq datasets were used to create a highly complex mixture?

<p>11</p> Signup and view all the answers

What was the purpose of filtering SNPs using bcftools filter?

<p>To ensure a minimum quality score (QUAL &gt; 80) and a minimum depth of coverage (INFO/DP &gt; 20)</p> Signup and view all the answers

How many barcodes were randomly selected from each of the 11 datasets?

<p>500</p> Signup and view all the answers

What was the DeGoulash pipeline used for?

<p>To deconvolute the mixed reads from the 11 datasets</p> Signup and view all the answers

How many times was each barcode selection repeated?

<p>10</p> Signup and view all the answers

What tool was used to extract SNPs from the barcode selection?

<p>FreeBayes v1.3.1</p> Signup and view all the answers

What was the purpose of using a scRNA-seq dataset with a mixture of four individuals?

<p>To determine the genomic location of SNPs in scRNA-seq and scATAC-seq</p> Signup and view all the answers

What was done to the selected reads after they were mixed?

<p>They were deconvoluted using the DeGoulash pipeline</p> Signup and view all the answers

What software was used to determine bi-parental bio-geographic ancestry using autosomal ancestry-informative SNPs?

<p>STRUCTURE (v2.3.4)</p> Signup and view all the answers

How many major populations were used to determine bi-parental bio-geographic ancestry in the 1000 Genomes database?

<p>Five</p> Signup and view all the answers

What tool was used to determine chromosome haplogroups from Y-SNP data?

<p>Y-leaf 2.1</p> Signup and view all the answers

How was paternal biogeographic ancestry inferred from Y haplogroups?

<p>From the geographic distribution of the identified Y haplogroups using literature resources.</p> Signup and view all the answers

What database was used to determine MtDNA haplogroups from mtDNA SNP data?

<p>Haplogrep2</p> Signup and view all the answers

How was maternal bio-geographic ancestry inferred from mtDNA haplogroups?

<p>From the identified mtDNA haplogroups using population data stored in the EMPOP database</p> Signup and view all the answers

What tool was used to calculate likelihood ratios (LRs) for individual genetic identification?

<p>De-goulash</p> Signup and view all the answers

What pipeline was used for mixture deconvolution in the scATAC-seq analysis?

<p>De-goulash pipeline</p> Signup and view all the answers

Study Notes

Single-cell DNA Sequencing for Mixture Deconvolution

  • Non-targeted single-cell DNA sequencing (scDNA-seq) was applied to address challenges of mixture deconvolution in forensic context.
  • scATAC-seq (Assay for Transposase-Accessible Chromatin with sequencing) technique was used to deconvolute complex blood mixtures.
  • scATAC-seq, together with De-goulash pipeline, can deconvolute mixtures of five individuals from both sexes with varying biogeographic ancestries.

Mixture Deconvolution and Genetic Characterization

  • Correct genetic characterization of biological sex and biogeographic ancestry of each mixture contributor was achieved.
  • Individual identity was established by analyzing scATAC-seq data mixtures.
  • Successful individual-specific mixture deconvolution was demonstrated for:
    • Highly complex mixtures of 11 individuals.
    • Balanced mixtures containing as few as 20 cells (10 per each individual).
    • Imbalanced mixtures with a ratio as low as 1:80.

Comparison with scRNA-seq

  • scDNA-seq detects more SNPs from fewer cells, providing higher sensitivity, which is valuable in forensic genetics.
  • scDNA-seq is more suitable for mixture deconvolution and individual identification compared to scRNA-seq.

Methodology

  • Autosomal SNPs were used for bi-parental bio-geographic ancestry determination using STRUCTURE and 1000 Genomes database.
  • Chromosome haplogroups were determined from Y-SNP data using Y-leaf.
  • MtDNA haplogroups were determined from mtDNA SNP data using Haplogrep2.
  • Individual genetic identification was calculated using likelihood ratios (LRs) in De-goulash.

In-silico Mixture Preparation and Analysis

  • In-silico balanced mixtures were created using publicly available single-individual scATAC-seq datasets.
  • Mixture deconvolution was performed using the De-goulash pipeline.
  • Individual genetic identification could not be performed from in-silico mixtures due to lack of reference data.
  • Correct individual-specific mixture deconvolution was assessed based on barcodes known to belong to one individual or the other.

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Deconvoluting Multi-Person Biological Mixtures PDF

Description

Learn about the application of single-cell DNA sequencing in forensic science to deconvolute complex blood mixtures using scATAC-seq technique and De-goulash pipeline.

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