Question: Insert Size Metrics of ATAC-Seq data
1
gravatar for Bioinfo_2006
7 weeks ago by
Bioinfo_200690
Bioinfo_200690 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 • 306 views
ADD COMMENTlink modified 7 weeks ago by igor8.1k • written 7 weeks ago by Bioinfo_200690

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 7 weeks ago by RamRS23k
2
gravatar for igor
7 weeks ago by
igor8.1k
United States
igor8.1k 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 7 weeks ago • written 7 weeks ago by igor8.1k

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

ADD REPLYlink written 5 weeks ago by Bioinfo_200690
1
gravatar for ATpoint
7 weeks ago by
ATpoint21k
Germany
ATpoint21k 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 7 weeks ago • written 7 weeks ago by ATpoint21k

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 7 weeks ago by Bioinfo_200690

Can you show samtools idxstats PBL_927-post_NR_aln_sorted_noM.bam?

ADD REPLYlink modified 7 weeks ago • written 7 weeks ago by ATpoint21k

enter image description here

ADD REPLYlink written 7 weeks ago by Bioinfo_200690

Removing MT now and checking again.

ADD REPLYlink written 7 weeks ago by Bioinfo_200690

grep -v 'MT' is what you need

ADD REPLYlink written 7 weeks ago by ATpoint21k

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 7 weeks ago by Bioinfo_200690

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 7 weeks ago by ATpoint21k

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 7 weeks ago by Bioinfo_200690

Insertsize metric plot still looks the same

enter image description here

ADD REPLYlink modified 7 weeks ago • written 7 weeks ago by Bioinfo_200690
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