Closed ck-theory closed 1 year ago
For anyone with the same issue - this key error is because the reads used in the pbmm2 align step are HiFi reads with kinetics, not subreads. So when they are aligned, there is important data missing. The two ways around this are to either re-generate subreads on a previously demultiplexed run or to run ccs-kinetics-bystrandify on your HiFi reads to reconstitute the subread data. With the help of the PB support team, below is the current workflow to avoid re-running lima:
conda install -c bioconda pbtk
inputs:
conda activate pbbam-2.1.0 ccs-kinetics-bystrandify inreads.bam out.kinetics.bam conda deactivate
conda activate smrtlink_11.0.0.146107 pbvalidate out.kinetics.bam pbmm2 align --sort out.kinetics.bam reference.fasta out.alignment.bam pbvalidate out.alignment.bam pbindex out.alignment.bam ipdSummary -j 20 out.alignment.bam --reference reference.fasta --gff out.gff --csv out.csv --bigwig out.bigwig
For anyone with the same issue - this key error is because the reads used in the pbmm2 align step are HiFi reads with kinetics, not subreads. So when they are aligned, there is important data missing. The two ways around this are to either re-generate subreads on a previously demultiplexed run or to run ccs-kinetics-bystrandify on your HiFi reads to reconstitute the subread data. With the help of the PB support team, below is the current workflow to avoid re-running lima:
Modification to include conversion of HiFi data to pseudo subread data
conda install -c bioconda pbtk
inputs:
reference.fasta
reference.fasta.fai inreads.bam (dmuxed hifi reads) conda activate pbbam-2.1.0 ccs-kinetics-bystrandify inreads.bam out.kinetics.bam conda deactivate
conda activate smrtlink_11.0.0.146107 pbvalidate out.kinetics.bam pbmm2 align --sort out.kinetics.bam reference.fasta out.alignment.bam pbvalidate out.alignment.bam pbindex out.alignment.bam ipdSummary -j 20 out.alignment.bam --reference reference.fasta --gff out.gff --csv out.csv --bigwig out.bigwig
Hi ck-theory
How did you install the smrtlink v11 with conda? I only see a v10 installation: https://anaconda.org/hcc/smrtlink-tools
Thank you
Hello, can you please help with the following error I have hit while running ipdSummary from smrtlink_11.0.0.146107 on data from a Sequel IIe demultiplexed microbial run with HiFi reads and Kinetics enabled? {SAMPLE and RUNID} have been redacted to protect customer data.
SMRTLink 11.0 Command Log
Methylation from HiFi-ASM assembled data
1) Index the fasta file
dataset create --generateIndices fasta.referenceset.xml {SAMPLE}.HiFiASM.assembly.fasta
2) Align the assembly to the PB data
pbmm2 align fasta.referenceset.xml {RUN_ID}.bc2014--bc2014.consensusreadset.xml --preset HiFi out.consensusalignmentset.xml
3) Call the methylation profiler
export SMRT_CHEMISTRY_BUNDLE_DIR=/data/programs/miniconda3/envs/smrtlink_11.0.0.146107/smrttools_install/userdata/chemistry/chemistry-pb-active
ipdSummary out.bam --reference {SAMPLE}.HiFiASM.assembly.fasta --gff out.gff --csv out.csv --bigwig out.bigwig