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virajbdeshpande / AmpliconArchitect

AmpliconArchitect (AA) is a tool to identify one or more connected genomic regions which have simultaneous copy number amplification and elucidates the architecture of the amplicon. In the current version, AA takes as input next generation sequencing reads (paired-end Illumina reads) mapped to the hg19/GRCh37 reference sequence and one or more regions of interest. Please "watch" this repository for improvements in runtime, accuracy and annotations for GRCh38 human reference genome coming up soon.
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The SV view: PDF file could not create different arcs! #125

Closed ZHONGHONGXIN closed 2 years ago

ZHONGHONGXIN commented 2 years ago

Dear AmpliconArchitect Team, l running AA to create a PDF file that displays the underlying sequence signatures of the amplicon, but my PDF file does not contain different arcs, and my command is as follows: /software/AmpliconArchitect/src/AmpliconArchitect.py --bed seed_50kb.bed \ --bam ncih520_pe_q0_redup.bam \ --sensitivems False \ --out ncih520 --ref GRCh38

l don't know how to adjust the parameter, to display different arcs, could you please help me realize it?

Best, HongXin

jluebeck commented 2 years ago

Hi HongXin,

Can you clarify if you ran into a runtime error or if you are reporting that your sample did not show any SV arcs in the visualization? If the later, do they show up in the .png? What aligner did you use to generate the bam file?

Jens

ZHONGHONGXIN commented 2 years ago

Hi HongXin,

Can you clarify if you ran into a runtime error or if you are reporting that your sample did not show any SV arcs in the visualization? If the later, do they show up in the .png? What aligner did you use to generate the bam file?

Jens

Hi Jens,

My situation is the latter, and the .png also doesn’t exist in any SV arcs. l choose BWA-MEM2 to generate the bam file. My aligner command is below:

  1. bwa-mem2 mem -t 30 -M hg38.fa |grep -E -v "chrM|random|chrUn_" | samtools view -@ 20 -m 3G -F 1804 -f 2 -b -o ${out}_pe.bam
  2. samtools sort -@ 30 ${out}_pe.bam -o ${out}_pe_sort.bam
  3. sambamba markdup -r -t 30 ${out}_pe_sort.bam ${out}_pe_q0_redup.bam

and my PDF file as shown: image

HongXin

jluebeck commented 2 years ago

Hi HongXin,

The BWA MEM -M flag will mark split alignments as duplicates, and they will be ignored by AA. Please remove that flag and re-align. The samtools view -f 2 flag is also problematic since it may filter anything that is not a proper pair (SVs are not properly paired reads). The samtools view -F 1804 flag may also cause issues since (among other things) it filters alignments which are not the primary alignment.

Jens

ZHONGHONGXIN commented 2 years ago

Hi HongXin,

The BWA MEM -M flag will mark split alignments as duplicates, and they will be ignored by AA. Please remove that flag and re-align. The samtools view -f 2 flag is also problematic since it may filter anything that is not a proper pair (SVs are not properly paired reads). The samtools view -F 1804 flag may also cause issues since (among other things) it filters alignments which are not the primary alignment.

Jens

Hi Jens, Thanks for your reply so fast, l accept your advice and try again.

HongXin