Comparison of variations detection between whole-genome amplification methods used in single-cell resequencing

Abstract

BackgroundSingle-cell resequencing (SCRS) provides many biomedical advances in variations detection at the single-cell level, but it currently relies on whole genome amplification (WGA). Three methods are commonly used for WGA: multiple displacement amplification (MDA), degenerate-oligonucleotide-primed PCR (DOP-PCR) and multiple annealing and looping-based amplification cycles (MALBAC). However, a comprehensive comparison of variations detection performance between these WGA methods has not yet been performed.ResultsWe systematically compared the advantages and disadvantages of different WGA methods, focusing particularly on variations detection. Low-coverage whole-genome sequencing revealed that DOP-PCR had the highest duplication ratio, but an even read distribution and the best reproducibility and accuracy for detection of copy-number variations (CNVs). However, MDA had significantly higher genome recovery sensitivity (~84 %) than DOP-PCR (~6 %) and MALBAC (~52 %) at high sequencing depth. MALBAC and MDA had comparable single-nucleotide variations detection efficiency, false-positive ratio, and allele drop-out ratio. We further demonstrated that SCRS data amplified by either MDA or MALBAC from a gastric cancer cell line could accurately detect gastric cancer CNVs with comparable sensitivity and specificity, including amplifications of 12p11.22 (KRAS) and 9p24.1 (JAK2, CD274, and PDCD1LG2).ConclusionsOur findings provide a comprehensive comparison of variations detection performance using SCRS amplified by different WGA methods. It will guide researchers to determine which WGA method is best suited to individual experimental needs at single-cell level.Electronic supplementary materialThe online version of this article (doi:10.1186/s13742-015-0068-3) contains supplementary material, which is available to authorized users.

Next-generation Sequencing (NGS) Analysis on Single Circulating Tumor Cells (CTCs) with No Need of Whole-genome Amplification (WGA)

“…Major chemistries employed in the singlecell WGA include the degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), the multiple displacement amplification (MDA), the multiple annealing and looping-based amplification cycles (mALBAC) and the ligationmediated PCR following a site specific DNA digestion as in the case of this study (4,17,19,20,(25)(26)(27). However, besides the operator capability, all these strategies are labor-intensive, require long time of execution and may imply different degrees of errors due to inappropriate starting in the early stages of amplification, or due to the specific chemicals used in the reaction (17,18,28).…”

The comparison of the performance of four whole genome amplification kits on ion proton platform in copy number variation detection

“…There are plenty of commercial WGA kits and different kits are based on different strategies: multiple displacement amplification (MDA), degenerate-oligonucleotide-primed PCR (DOP-PCR), and multiple annealing and looping-based amplification cycles (MALBAC, MALBAC-like) are three major WGA strategies. Different strategy may cause different WGA performance and some studies have paid efforts in the comparison of WGA methods [15][16][17][18][19]. However, current related researches are conducted on Illumina sequencing platforms, the results have not validated on Ion Proton platform.…”

Robust high-performance nanoliter-volume single-cell multiple displacement amplification on planar substrates

“…Of these, three WGA strategies have been most widely adopted, each with its relative strengths and weaknesses (10,11): PCR-based protocols (1,12), multiple displacement amplification (MDA) (2-4, 7), and combinations of displacement preamplification followed by PCR (8,(13)(14)(15). Of these strategies, MDA is most widely used when…”