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Output from Minimap2 Loaded into IGV - Blank? #187

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MerillaM opened this issue Jun 19, 2018 · 5 comments
Closed

Output from Minimap2 Loaded into IGV - Blank? #187

MerillaM opened this issue Jun 19, 2018 · 5 comments

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@MerillaM
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Hello everyone, I'm having trouble visualizing my PacBio data in IGV, as it looks like there is no coverage / the .bam files as input are blank.

Starting with subreads.bam data, I went through CCS, Classify, Cluster, Minimap2, Conversion to .bam, sort, and index, before loading into IGV. My command line prompts for each of these steps are below. Can anyone tell me why my coverage in IGV would be blank? The files aren't empty, but nothing shows up. I'm wondering if I made a mistake somewhere in the middle that would have corrupted the future .bam files, or something like that. No error messages were produced during any of these steps. Thanks in advance!

CCS Step:
ccs --noPolish --minLength=300 --minPasses=1 --minZScore=-9999 --maxDropFraction:0.8 --minPredictedAccuracy=0.9 --minSnr=4 180938.subreads.bam CCS_180938.bam

dataset create --type ConsensusReadSet CCS_180938.xml CCS_180938.bam

Classify Step:
pbtranscript classify CCS_180938.xml 180938_isoseq_draft.fasta --flnc=180938_isoseq_flnc.fasta --nfl=180938_isoseq_nfl.fasta

Cluster Step:
pbtranscript cluster 180938_isoseq_flnc.fasta 180938_polished_clustered.fasta --quiver --nfl_fa=180938_isoseq_nfl.fasta --bas_fofn=scratch/PACBIO/Merilla_PACBIO/working/consensus_reads/xml/subreadsets/180938.subreadset.xml

Minimap2 Step:
minimap2 -t 5 -ax map-pb hg38.fasta ./cluster_out/all_quivered_hq.100_30_0.99.fasta > 180938.fasta.sam

Convert to BAM, Sort, and Index Step:
samtools view -Sb 180938.fasta.sam > 180938.fasta.bam
samtools sort 180938.fasta.bam > 180938.fasta.sorted.bam
samtools index 180938.fasta.sorted.bam
@lh3
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lh3 commented Jun 19, 2018

Is your final bam 180938.fasta.sorted.bam empty? If not, you should ask IGV developers.

@MerillaM
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@lh3 It isn't empty. I will do that, thank you!

@wdecoster
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wdecoster commented Jun 19, 2018 •
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@MerillaM apologies if you already checked this, but have you tried zooming? Is the right reference genome loaded in IGV? Are you in a location in which you expect reads, e.g. check the bam first using samtools view?

@MerillaM
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@wdecoster I did zoom in and it's the correct genome. I checked samtools view, and that did the trick! There isn't a lot of data, but I was also not looking in the right spot. Thanks so much!

@qzhaojing
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@MerillaM
the question is the .sam file the minimap generated. it is not a .sam/bam file similar to bwa. so the solution is to use .paf file. when use minimap2, you can first export .paf file , and use
paftools.js splice2bed convert spliced alignment in PAF/SAM to BED12
in minimap2 convert paf file to .bed.
and now, u can import the .bed to IGV

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@lh3 @wdecoster @qzhaojing @MerillaM