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?
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What reference genome was used to set the reference alleles in PLINK v2.00?
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Which tool was used to overlap the identity SNP lists obtained from scATAC-seq data with the WES and GSA reference data?
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Why was individual genetic identification not possible from in-silico mixture?
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How were the correct individual-specific mixture deconvolutions assessed?
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What specific technique was used in this study to perform non-targeted single-cell DNA sequencing (scDNA-seq)?
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What is the significance of separating cells from biological mixtures in forensic science?
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What was the complexity of the blood mixtures tested in this study?
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What are the two main methods of single-cell DNA analysis mentioned in the text?
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What key information was successfully extracted from the deconvoluted mixture samples?
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Explain the advantage of scDNA-seq over scRNA-seq in terms of SNP detection.
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Describe the in-silico experiments conducted to further test the efficacy of the approach.
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What is the potential application of compound markers (two or more autosomal SNPs) in forensic genetics?
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What is the significance of the in-silico results regarding mixture complexity?
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What is a major limitation of using compound markers for forensic purposes?
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What data analysis pipeline was used in this study to deconvolute the single-cell DNA sequencing data?
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What is the overall conclusion of this study regarding the use of scDNA-seq, specifically scATAC-seq, in forensic applications?
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What is the significance of the date "Received 22 August 2023" in the context of the research article?
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Explain the potential impact of this study on forensic science.
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What is the meaning of the CC BY license mentioned in the article?
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Identify the two departments at Erasmus MC University Medical Center Rotterdam that are associated with the authors of the research article.
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How many single-individual scATAC-seq datasets were used to create a highly complex mixture?
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What was the purpose of filtering SNPs using bcftools filter?
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How many barcodes were randomly selected from each of the 11 datasets?
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What was the DeGoulash pipeline used for?
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How many times was each barcode selection repeated?
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What tool was used to extract SNPs from the barcode selection?
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What was the purpose of using a scRNA-seq dataset with a mixture of four individuals?
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What was done to the selected reads after they were mixed?
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What software was used to determine bi-parental bio-geographic ancestry using autosomal ancestry-informative SNPs?
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How many major populations were used to determine bi-parental bio-geographic ancestry in the 1000 Genomes database?
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What tool was used to determine chromosome haplogroups from Y-SNP data?
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How was paternal biogeographic ancestry inferred from Y haplogroups?
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What database was used to determine MtDNA haplogroups from mtDNA SNP data?
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How was maternal bio-geographic ancestry inferred from mtDNA haplogroups?
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What tool was used to calculate likelihood ratios (LRs) for individual genetic identification?
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What pipeline was used for mixture deconvolution in the scATAC-seq analysis?
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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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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.