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Genotyping amplicons from SARS-CoV-2 viruses to verify the identity of PCR products

ABSTRACT: Genotyping amplicons from SARS-CoV-2 viruses to verify the identity of PCR products

PROVIDER: PRJEB83339 | ENA |

REPOSITORIES: ENA

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Project description:Highly specific amplification of complex DNA pools without bias or template-independent products (TIPs) remains a challenge. We have developed a procedure using phi29 DNA polymerase and trehalose and optimized control of amplification to create micrograms of specific amplicons without TIPs from down to sub-femtograms of DNA. The amplicons from 5 ng and 0.5 ng DNA, which were from originally good quality of gDNA (05-050), or partially degraded gDNA (04-018), faithfully demonstrated all previously known heterozygous segmental duplications and deletions (3 Mb to 18 kb) located on chromosome 22 and even a homozygous deletion smaller than 1 kb with high resolution chromosome-wide CGH. Specifically, HR-CGH with 5 ng-input gDNA-derived amplicon detected all previously known chromosomal segmental aberrations in chromosome 22 in samples from two different probands, and was indistinguishable from the HR-CGH result with native gDNA from the same probands (Fig. 4, Fig. S3 and S4). The break points were also precisely demonstrated. These include a heterozygous genomic segmental duplication (3 copies, 3 Mb in size, sample 05-050, Fig. 4) and 2 different heterozygous deletions (1 copy, 1.4 Mb and 18 kb respectively, sample 04-018, Fig. S4), all of which are located in or bounded by regions of low copy repeats (LCRs). In addition, a previous known homozygous deletion of 975 bp (in 04-018 and 05-050) was again accurately demonstrated (05-050 data showed in Fig. 4c), although sometimes (04-018) the data was a little noisier than with unamplified DNA (Fig. S4d). In contrast, the Wpa-40oC resulted in abundant signal noise and failed in detection of these copy number aberrations (Fig. 4, Fig. S3, S4). Impressively, HR-CGH with 0.5 ng gDNA-derived amplicons via Wpa also clearly detected the known CNVs, although noisier (Fig. S4). The 0.1ng gDNA derived amplicons via Wpa could not unambiguously show CNVs because of higher variability of signals, but the CNVs’ patterns were mostly well maintained (Fig. S4 for 04-018). We did also notice some locus-imbalance in the amplicon, however this was minimized, and was reproducible when the input was above a certain threshold amount, and it could be well compensated if the same amplified reference sample was applied in parallel as showed above. Keywords: Whole-pool amplification, high resolution comparative genome hybridization (HR-CGH)

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Genotyping amplicons from SARS-CoV-2 viruses to verify the identity of PCR products

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