Comparison of Whole Genome Sequencing Effects of Group A Rotavirus Based on Multiplex RT-PCR Amplification and Double Stranded cDNA Synthesis
To establish a single tube multiplex reverse transcription-polymerase chain reaction(RT-PCR)amplification method that targeted the whole genome of Group A rotavirus(RVA),and to compare the effect with the double stranded cDNA synthesis method applying in the next generation sequencing of RVA clinical specimens,nucleic acid was extracted from 18 RVA-positive fecal samples of hospitalized children with diarrhea from a monitoring hospital in 2022.The whole genome of RVA was amplified using a single tube multiplex RT-PCR method,and the double stranded cDNA synthesized after the RNA was reverse transcripted with random primers.The Illumina platform was used for next generation sequencing of these two different types of products.CLC software was used to map the sequencing data,and analyze the effective data amount,sequencing depth,and whole genome coverage.The IGV software was used to view the sequencing coverage trace throughout the whole genome.The effective data amount of multiplex RT-PCR amplification products sequencing and double-stranded cDNA sequencing were 69.39％～99.83％and 0.38％～92.99％,respectively.The average sequencing depth was 10172×～68156× and 35×～61783 ×,respectively,and the whole genome coverage of sequencing depth of more than 10× was 98.93％～100％and 86.28％～100％,respectively.From the perspective of whole genome sequencing depth,the multiplex RT-PCR amplification products sequencing was relatively uniform in the depth of the same gene segment and quite different among the 11 segments.However,the double-stranded cDNA sequencing showed a uniform depth between different gene segments,but the sequencing depth near the 5'and 3'ends of the same segment was lower.The nucleotide sequences obtained by both methods were basically consistent,some gene segments showed 1～2 different nucleotide sites within the 100bp region near the 5'or 3'end.Due to the specificity of primers among different genotypes,the multiplex RT-PCR efficiency need to be further verified for rotavirus types that was not covered in this study.Although the one-tube multiplex RT-PCR amplification method and the double-stranded cDNA synthesis method have their own advantages and disadvantages,they are both suitable for the promotion of next generation sequencing of RVA whole genome at the primary institution.
Group A rotavirusWhole genome sequencingMultiplex RT-PCR amplificationAmplicon sequencingDouble stranded cDNA synthesis
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