TODO: mapping virus reads should use TopHat2, BWA or Bowtie2 could not detect splice events.

RNA sequencing (RNA-seq)

• Library preparation for strand specific RNA sequencing was carried out using the NEXTflex Directional RNA-Seq Kit (Bioo Scientific) according to the manufacturer’s instructions.
• Libraries were sequenced on the Illumina HiSeq 2500 platform.
• To allow detection of splice events that extend over the origin, reads were mapped to two concatenated copies of the MCVSyn or MCVSyn-hpko genomes using TopHat2 v 2.0.13 [94].
• The positions of mapped reads and junctions were subsequently collapsed back on unit-length genomes.
• From the resulting SAM files, we counted the number of unspliced reads that extended over splice sites of junctions detected by TopHat to determine splice site efficacy and frequency of individual junctions.
• To estimate transcript abundance, for each combination of splice junctions that mapped within either the major early or late transcription cassettes we calculated a relative strand-specific combinatorial frequency value by multiplying observed frequency values for individual donor sites.

• The relative ratio of late to early transcripts was subsequently estimated by calculation of normalized RPKM (reads per kilobase per million mapped reads) for each of the transcripts.

• Based on the Juliane paper, "A Comprehensive Analysis of Replicating Merkel Cell Polyomavirus Genomes Delineates the Viral Transcription Program and Suggests a Role for mcv-miR-M1 in Episomal Persistence," particularly in the "RNA sequencing (RNA-seq)" methods section, they used strand-specific RNA sequencing. This explains why they are able to separate the coverage into blue (positive strand) and red (negative strand). The original text reads: "Library preparation for strand-specific RNA sequencing was carried out using the NEXTflex Directional RNA-Seq Kit (Bioo Scientific) according to the manufacturer’s instructions."

• Unfortunately, your RNA-seq data is not strand-specific. Additionally, the primary focus of your sequencing is on the human genome, while the virus is a by-product, meaning we only have a very limited number of virus reads. For example, on average, there are only about 10 reads for VP1 and VP2 per sample. For this reason, creating a plot similar to the one in Juliane's paper, with separate coverage for positive and negative strands, is not feasible.

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