Question: Insert Size Metrics of ATAC-Seq data
1
gravatar for Bioinfo_2006
12 months ago by
Bioinfo_2006140
Bioinfo_2006140 wrote:

I have paired-end sequencing data from ATAC-Seq and the fastq files had nextera adaptors. So I trimmed them the following way:

trim_galore --paired --nextera ${names[${SLURM_ARRAY_TASK_ID}]}_R1.fastq ${names[${SLURM_ARRAY_TASK_ID}]}_R2.fastq

Then did Bowtie

bowtie2 -p 4 -q --very-sensitive  -x genome -1 ${names[${SLURM_ARRAY_TASK_ID}]}_R1_val_1.fq -2 ${names[${SLURM_ARRAY_TASK_ID}]}_R2_val_2.fq -S "${names[${SLURM_ARRAY_TASK_ID}]}"_aln_unsorted.sam

While doing ATAC-Seq insert size plotting, I see blue region in the histogram corresponding to flipped reads. Could someone point out where I went wrong? Please see below histogram, it looks the same for all samples before filtering for Mt reads, and I also tried filtering out MT reads and PCR duplicates, still looks the same.

Before removing mitochondrial reads

After filtering mito and PCR duplicates

atac-seq picard • 860 views
ADD COMMENTlink modified 12 months ago by igor11k • written 12 months ago by Bioinfo_2006140

Please see How to add images to a Biostars post to add your images properly. You need the direct link to the image, not the link to the webpage that has the image embedded (which is what you have used here)

ADD REPLYlink written 12 months ago by RamRS27k
2
gravatar for igor
12 months ago by
igor11k
United States
igor11k wrote:

Are your reads 75bp? That seems to be where the switch happens. If your fragments are shorter than your reads, Bowtie would consider those discordant. From the manual:

Bowtie 2’s default behavior is to consider overlapping and containing as being consistent with concordant alignment. By default, dovetailing is considered inconsistent with concordant alignment.

As a test, you can try trimming your initial reads to 35bp (much shorter but still mostly mappable) and see how that changes the result.

ADD COMMENTlink modified 12 months ago • written 12 months ago by igor11k

Thanks so much for pointing this out, trimming to 35bp solved the problem.

ADD REPLYlink written 11 months ago by Bioinfo_2006140
1
gravatar for ATpoint
12 months ago by
ATpoint36k
Germany
ATpoint36k wrote:

Did you remove mitochondrial reads? If not, do so. I bet money this blue part will disappear.

samtools idxstats your.bam \
  | cut -f1 \
  | grep -v 'chrM' \
  | xargs samtools view -o your_filtered.bam your.bam

Depending on your chromosome names, you might need to set 'chrM' to something else.

ADD COMMENTlink modified 12 months ago • written 12 months ago by ATpoint36k

Edited my post. Forgot to include that I tried that as well. I feel it has something to do with trimming adapters, and the alignment afterwards, that is why posted the code to check if am missing parameters.

ADD REPLYlink written 12 months ago by Bioinfo_2006140

Can you show samtools idxstats PBL_927-post_NR_aln_sorted_noM.bam?

ADD REPLYlink modified 12 months ago • written 12 months ago by ATpoint36k

enter image description here

ADD REPLYlink written 12 months ago by Bioinfo_2006140

Removing MT now and checking again.

ADD REPLYlink written 12 months ago by Bioinfo_2006140

grep -v 'MT' is what you need

ADD REPLYlink written 12 months ago by ATpoint36k

It is strange. I tried

samtools idxstats PBL_927-post_NR_aln_sorted.bam | cut -f1 | grep -v 'MT' | xargs samtools view -b PBL_927-post_NR_aln_sorted.bam -o PBL_927-post_NR_aln_sorted_noM.bam

as well as

samtools idxstats PBL_927-post_NR_aln_sorted.bam | cut -f1 | grep -v 'MT' | xargs samtools view -b PBL_927-post_NR_aln_sorted.bam -o PBL_927-post_NR_aln_sorted_noM.bam

but unable to remove the MT sequences.

ADD REPLYlink written 12 months ago by Bioinfo_2006140

I am using this command in my work routinely and it works so it must be something on your side, maybe an old samtools version, I unfortunately cannot tell from here. Try updating samtools.

ADD REPLYlink written 12 months ago by ATpoint36k

I know its the same thing I have been using for other data, so it is really frustrating. Am sure it is something small that am missing out. I will check this and update the post.

ADD REPLYlink written 12 months ago by Bioinfo_2006140

Insertsize metric plot still looks the same

enter image description here

ADD REPLYlink modified 12 months ago • written 12 months ago by Bioinfo_2006140
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