Abstract
Single Gene Disorders (SGD) are still routinely diagnosed using PCR-based assays that need to be developed and validated for each individual disease-specific gene fragment. The TruSight One sequencing panel currently covers 12 Mb of genomic content, including 4813 genes associated with a clinical phenotype. When only a limited number of cells are available, whole genome amplification (WGA) is required prior to DNA target capture techniques such as the TruSight One panel. In this study, we compared 4 different WGA methods in combination with the TruSight One sequencing panel to perform single nucleotide polymorphism (SNP) genotyping starting from 3 micro-manipulated cells. This setting simulates clinical settings such as day-5 blastocyst biopsy for Preimplantation Genetic Testing (PGT), liquid biopsy of circulating tumor cells (CTCs) and cancer-cell profiling. Bulk cell samples were processed alongside these WGA samples to serve as a performance reference. Target coverage, coverage uniformity and SNP calling accuracy obtained using any of the WGA, is inferior to the results obtained on bulk cell samples. However, results after REPLI-g come close. Compared to the other WGA methods, the method using REPLI-g WGA results in a better coverage of the targeted genomic regions with a more uniform read depth. Consequently, this method also results in a more accurate SNP calling and could be considered for clinical genotyping of a limited number of cells.
Highlights
Several clinical settings such as Preimplantation Genetic Testing (PGT) [1], liquid biopsy of circulating tumor cells (CTCs) and cancer-cell profiling of tiny biopsies could benefit from a method allowing genotyping of large panels of single nucleotide polymorphism (SNP), starting with only a few cells as input
SNP calling performance of these whole genome amplification (WGA) methods in combination with target capture panels has been studied [19,22,23]: Babayan et al studied the accuracy of SNP/mutation, indel, and copy number aberrations (CNA) calling after exome sequencing of Ampli1, REPLIg, and PicoPlex WGA products [19]
As WGA methods based on multiple displacement amplification (MDA) are reportedly [9] better suited for SNP detection because they use the highfidelity phi29 polymerase and amplify more regions of the genome compared to PCR-based methods, it is to be expected that REPLI-g would outperform MALBAC, SurePlex and Ampli1 in this study
Summary
Several clinical settings such as PGT [1], liquid biopsy of CTCs and cancer-cell profiling of tiny biopsies could benefit from a method allowing genotyping of large panels of SNPs, starting with only a few cells as input. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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