Hello, Could you please advise on the following:
We have ChIP-seq data with paired-end Illumina reads. For some of the samples only about 11% or reads could be mapped with Bowtie. When remapping these samples with Bowtie2, up to 85% reads could be mapped, but the pairs have been lost, meaning that for most mapped reads there is no pair available. What could go wrong and how to fix it? Thanks!
1 answer
The numbers of reads is the same, but their quality seems to be different: I have mapped with Bowtie separately each of the two paired fastq files: for one file I've got 50% reads with at least one reported alignment, whereas for the second fastq file I've got 31% reads with at least one reported alignment. I guess this explains how I end up with even smaller percent of aligned pairs when using paired-end alignment. This is then unrelated to the insert size... But how to fix this is the question
Have a look at the quality of read 1 and read2 with FASTQC or similar (fastp ). Do the quality values of the second read drop off markedly along the read ? Try trimming ? Or as suggested above BWA. Bad R2 is pretty common, especially on >150bp reads from some illumina sequencers.
Log in to answer this question.
-
Bowtie paired-end mapping: insert size issue
written by biostart
-
ChIP-Seq. plotFingerprint interpretation: 2 ChIPs are like IgG control. Conclusions?
written by KB* •
-
HOMER works incorrectly with -sspe option for paired-end reads
written by biostart
-
Speed up Tophat2 running
written by yuabrahamliu •
-
No proper pairs for some files upon filtering using Bamtools
written by dolores •
-
Chip-seq mapping (using bowtie2 & tophat2) What's different ?
written by leenaehyeon •
-
Filtering high quality reads from bowtie2
written by yulia.gordon •
-
bowtie2 output all aligned reads from paired alignment (even unconcordant pairs or single reads)
written by JV
-
Best way to map paired-end, uniquely mapped reads with Bowtie2?
written by Ian
-
Almost Nothing Mapped Using Bwa Or Bowtie. But A Lot Of Mapping In Blast
written by lwc628
Try BWA with just one file, over even a subset of your reads. BWA will estimate insert size from the mapping, and its output may help you understand what went wrong with your Bowtie mapping. If you want to stick with Bowtie / Bowtie2, you then may use BWA estimated mean and sd insert size values as input for Bowtie.
I can estimate the average DNA fragment length as ~150 based on the reads which successfully aligned