GA2411 - scWGS kit evaluation: BioSkryb PTA vs Qiagen MDA on MM1S cells - Illumina Infinium GSAv3 data

<h2>Summary</h2><p dir="ltr">In this study, we systematically compared two single cell whole genome sequencing (scWGS) kits in the MDA-based REPLI-g Single Cell Kit from Qiagen and the PTA-based ResolveDNA Whole Genome Amplification Kit from BioSkryb. The kits were evaluated across several key metrics, including amplification bias and coverage uniformity and copy number variant (CNV) calling.</p><h2>Methods</h2><p dir="ltr">Cultured MM.1S cells (a multiple myeloma cell-line) were incubated with propidium iodide (PI) and through Fluorescence-Activated Cell Sorting (FACS) sorted using the BD FACSAria IIu. Single or multiple cells were deposited using single cell mode into 96-well plates containing the recommended buffer for the Qiagen Repli-G MDA and BioSkryb ResolveDNA PTA kits. Cell sorting was performed on the same occasion and from the same sample of MM1.S cells to ensure minimal sample variation. For each kit, 26 wells with a single cells and two wells with 10 cells were FACS sorted into the 96-well plate. In addition, two wells with a mini-bulk of 10 cells, two positive controls wells with 1ng of genomic DNA (provided with the BioyoSkryb kit) in buffer and two empty wells (negative control containing only with buffer) were also included in each plate. Whole Genome Amplification (WGA) was performed using Multiple Displacement Amplification (MDA) using the Repli-G kit (Qiagen, Cat no: 1124559, 180318 and 1124693) or Primary Template-directed Amplification (PTA) using the ResolveDNA kit (BioSkryb, PN: 100136, 100691 and 100940) was performed according to the manufacturer’s instructions. After amplificaiton, ten random single wells, one mini-bulk and one positive control well were further selected for library preparation, using the same BioSkryb or Qiagen kit and finally qPCR quantification prior to sequencing. Illumina sequencing data for the scWGS kit samples is available on ENA under the study accession PRJEB97702. The same WGA DNA was also subjected to genotyping using the Illumina Infinium Global Screening Array v3.0 (Illumina). Arrays were processed on an Illumina iScan system according to the manufacturer's instructions. Initial data processing and genotype calling were performed in GenomeStudio (Illumina, v2.0.3), using Human Reference Genome build 38 (GRCh38) as reference.</p><p dir="ltr"><b>Genotyping details</b></p><ul><li>Number of analyzed markers: 650 181</li><li>Number of analyzed samples or individuals: 22</li><li>Organism: Homo sapiens</li></ul><p dir="ltr">The genotyping was performed using the Illumina Infinium assay and the results were analyzed using the software GenomeStudio 2.0.3.</p><ul><li>BeadChip type: GSA-24v3-0_A2</li><li>Manifest file: GSA-24v3-0_A2.bpm</li><li>Genome build version: 38</li><li>Cluster file ICF: GSA-24v3-0_A1_ClusterFile.egt</li></ul><p dir="ltr">ICF (Illumina Cluster File) result files represent genotyping data generated using cluster definitions established by Illumina from a reference DNA sample set. These reference-based cluster files are used when project-specific cluster files (PCF) are not available—typically in projects with fewer than 100 samples, where creating reliable project-based clusters is not feasible. In such cases, Illumina recommends using ICF files as they provide standardized clustering information derived from a larger, validated reference dataset, ensuring consistent genotype calling across experiments.</p><p dir="ltr">The result folders and files have the extension name ̈PLUS ̈. This refers to that the file is based on genotype data exported from PLUS DNA strand according to the Human Reference Genome build38, as given in the marker file GSA-24v3-0_A2.csv.</p><h2>Contents</h2><p><br></p><p dir="ltr">Content are compressed using 7-Zip</p><p dir="ltr"><b>Folder: XK-4162_250519_ResultReport</b></p><ul><li>Description of result report: XK-4162_250519_ResultReport.pdf</li><li>Manifest file: GSA-24v3-0_A2.csv</li><li>Cluster file: GSA-24v3-0_A1_ClusterFile.egt</li></ul><p dir="ltr"><b>Folder: XK-4162_250519_ResultReport_ICF_PLUS</b></p><ul><li>Statistics from the genotyping: XK-4162_250519_GenotypingStatistics_ICF_PLUS.xlsx</li><li>Genotype data: XK-4162_250519_SNPGenotypeExport_ICF_PLUS.txt</li><li>Results from HapMap comparison: XK-4162_250519_HapMap_1KG_Comparision_ICF_PLUS.xlsx</li><li>Identity test: XK-4162_250519_Identify_Item.xlsx</li><li>Gender test: XK-4162_250519_GenderEstimation.xlsx</li></ul><p dir="ltr"><b>Folder: XK-4162_250519_PLINK_ICF_PLUS</b></p><p dir="ltr">PLINK files</p><ul><li>XK-4162_250519_PLINK_ICF_PLUS.map</li><li>XK-4162_250519_PLINK_ICF_PLUS.ped</li></ul><p dir="ltr"><b>Folder: XK-4162_250519_IDAT</b></p><ul><li>IDAT files</li><li>XK-4162_250519_Samplesheet.csv</li></ul><p><br></p>