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Question: BigWig file of strand-specific RNA-seq data
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Hi all, I began to process strand-specific RNA-seq data from dUTR method library these days. I added the flag --rna-strandness RF to the default hisat2 commandline on paired-end RNA-seq data and each line of the final bam file also got a XS:A:+ or XS:A:- value to indicate the plus or minus strand. Then, I coverted the bam files to bigwig files and submited them to UCSC browser to check the data. However, the reads in the browser still displayed no strand specificity. I mean, a read would be displayed by both plus and minus strand, rather than only one strand. (See the figure below). I feel confused and don't known what's wrong with that. Could any one give me some help? My code to covert bam to bigwig is like this: genomeCoverageBed -bg -split -ibam KO.bam -strand + -g mm10.chrom.sizes > KO.p.bedgraph genomeCoverageBed -bg -split -ibam KO.bam -strand '-' -g mm10.chrom.sizes > KO.m.bedgraph norm_bedgraph.pl -t 33441081 -i 'KO.p.bedgraph KO.m.bedgraph' -m 'KO.p.bedgraph' bedGraphToBigWig KO.p.bedgraph.normolized mm10.chrom.sizes KO.p.bw bedGraphToBigWig KO.m.bedgraph.normolized mm10.chrom.sizes KO.m.bw

reads were mapped to both plus and minus strands, not only one strand

ADD COMMENTlink 2.3 years ago yuabrahamliu • 40 • updated 2.3 years ago Devon Ryan 90k
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There is no concept of strand in bigWig files, they only hold positions with values associated with them. If you want to have strand-specific bigWig files then you'll need to make a bigWig file for each strand. You can do that with bamCoverage from deepTools, using the --filterRNAstrand option.

genomeCoverageBed is using a different understanding of strand than what you want. It's splitting individual reads by their orientation, so since you presumably have paired-end data, the mates from each pair will end up in different files. What you want instead is a tool that will treat mates the same and count them (or not) depending on the orientation of the fragment from which they arose.

ADD COMMENTlink 2.3 years ago Devon Ryan 90k
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Thank you so much for your help. I have changed genomeCoverageBed to bamCoverage, but unfortunately, the plus and minus strands still cannot be distinguished. I feel really confused, I don't know if there is something wrong with my code. Do you have any suggestions on dUTP data? Thank you. trimmomatic PE -threads 20 SRR_1.fastq SRR_2.fastq trimmed_1.fastq unpaired_1.fastq trimmed_2.fastq unpaired_2.fastq SLIDINGWINDOW:4:30 hisat2 -p 20 --rna-strandness RF -x mm10hisatidx -1 trimmed_1.fastq -2 trimmed_2.fastq |samtools sort -@ 20 > test.bam samtools index -@ 20 test.bam bamCoverage -p 20 --normalizeTo1x 2150570000 --filterRNAstrand forward -b test.bam -o test.m.bw bamCoverage -p 20 --normalizeTo1x 2150570000 --filterRNAstrand reverse -b test.bam -o test.p.bw enter image description here

ADD REPLYlink 2.3 years ago
yuabrahamliu
• 40
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Look at the scale, there is a clear distinction with presumably either some low level of anti-sense transcription or (more likely) the strandedness of the library isn't perfect.

ADD REPLYlink 2.3 years ago
Devon Ryan
90k

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