Picard-Tools Or Trimmomatic Flip Read From Fr Or Rf??

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10.5 years ago

mengqingying ▴ 30

I have some PE RNAseq reads contaminated with adapter sequences.

Before removing the adapters, i mapped the reads using bowtie2 -> samtools -> picard-tools "CollectInsertSizeMetrics.jar" and got histogram as shown in the first figure.

After removing the adapters with Trimmomatic, i again run bowtie2 -> samtools -> "CollectInsertSizeMetrics.jar" and got the 2nd histogram with many RF reads, as shown in blue color in the plot. Actually here i already set bowtie2 parameter --no-discordant to remove discordant reads.

I cannot figure out the reason there are so many reads flip their orientations from FR -> RF? I wonder if this change are caused by Trimmomatic, or picard? How this happened?! Any one can help?

Figure1_beforeQC Figure2_afterQC

rnaseq • 2.6k views

ADD COMMENT • link updated 10.5 years ago by Devon Ryan 104k • written 10.5 years ago by mengqingying ▴ 30

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Can't see the figure 2. Can you repost it?

ADD REPLY • link 10.5 years ago by Ashutosh Pandey 12k

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It was just a formatting issue, it should be fixed now.

ADD REPLY • link 10.5 years ago by Devon Ryan 104k

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