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high percentage of **total_single_sided_mapped** #122
In the results (for proximity ligations in the genome with the help of linker), I found a high percentage of total_single_sided_mapped ~30%, what could be the reason for this?
I identified the linker by using the tool ChIA-PET2 to extract the real reads from the raw data and remove the linker part before going to pairtools to sort the pairs
I usually use bwa aln instead of bwa mem for short reads mapping. It's syntax is more complicated, but it saved me a whole dataset once, when after trimming all the reads were 20-30 bps.
Quick assessment: for human genome you may expect unique hit of the read longer than 15 bps, below that value it may not help much to keep it, because short reads will have a large chance to be multi-mapped.
Let me know how it goes! I may share my version of distiller that uses bwa aln if that helps.
I split the data into 16-70bp and greater than 70bp, using bwa aln and bwa mem respectively, and found that there are ~30% of total_single_sided_mapped in the 16-70bp data. Also, ~20% of the total_single_sided_mapped reads still exist in the greater than 70bp data.
Hi!
Your procedure makes me think about the cases when the adapter sequence was clipped from one side of the read but not another (so that one side is short and another is normal). Would these cases fall into 16-70bp or >70bp category by your design?
It's also important that both R1 and R2 of the same read go into the same file for bwa mapping.
Other possibilities worthy checking:
fastqc after trimming will show if there are any undercut adapter sequences, or some other sequences are repeatedly shared between the reads
blast after extracting unmapped ones from .bam files would by help, it will provide an insight why some pieces of the reads are reported as unmapped by bwa: samtools view -b -f 4 HiC_057_439_embryo_rep1.lane1.XA.dm3-embryo.bam | samtools fasta --
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Phlya commentedon Apr 12, 2022
Hi, did you do anything special to deal with the linker sequence?
wbszhu commentedon Apr 12, 2022
I identified the linker by using the tool ChIA-PET2 to extract the real reads from the raw data and remove the linker part before going to pairtools to sort the pairs
wbszhu commentedon Apr 12, 2022
I can't quite answer the question about the experimental handling of the linker. Because an unpublished technique is involved.
agalitsyna commentedon Apr 12, 2022
Then, if the resulting read length is shorter than ~30 bps, bwa mem might struggle to map these reads. It's dedicated for long reads alignments only.
wbszhu commentedon Apr 13, 2022
Thanks，Do you think the resulting read length is shorter than ~30 bps should be kept?
agalitsyna commentedon Apr 13, 2022
I usually use
bwa alninstead ofbwa memfor short reads mapping. It's syntax is more complicated, but it saved me a whole dataset once, when after trimming all the reads were 20-30 bps.Quick assessment: for human genome you may expect unique hit of the read longer than 15 bps, below that value it may not help much to keep it, because short reads will have a large chance to be multi-mapped.
Let me know how it goes! I may share my version of distiller that uses
bwa alnif that helps.wbszhu commentedon Apr 13, 2022
Thanks for your sharing, I will have a try.
wbszhu commentedon Apr 18, 2022
I split the data into 16-70bp and greater than 70bp, using bwa aln and bwa mem respectively, and found that there are ~30% of total_single_sided_mapped in the 16-70bp data. Also, ~20% of the total_single_sided_mapped reads still exist in the greater than 70bp data.
agalitsyna commentedon Apr 18, 2022
Hi!
Your procedure makes me think about the cases when the adapter sequence was clipped from one side of the read but not another (so that one side is short and another is normal). Would these cases fall into 16-70bp or >70bp category by your design?
It's also important that both R1 and R2 of the same read go into the same file for bwa mapping.
Other possibilities worthy checking:
fastqcafter trimming will show if there are any undercut adapter sequences, or some other sequences are repeatedly shared between the readssamtools view -b -f 4 HiC_057_439_embryo_rep1.lane1.XA.dm3-embryo.bam | samtools fasta --