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
Thank you so much for your help. I have changed
genomeCoverageBed
tobamCoverage
, 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
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.