Aligning PacBio hifiasm CCS to reference genome using pbmm2
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5 weeks ago
Lu K • 0

Hi,

I had consensus reads (ccs.bam) from a PacBio sequencing. I used the assembler hifiasm to create an assembly on these CCS. I got five .gfa files. I have transformed .gfa file using bandage and awk command. Contigs from de novo assemblies (.fasta file) were used in standalone blast against reference genome. I have the coordinates which mapped to the reference genome.

I am interested to align .fasta and reference genome using pbmm2

A. Generate index file for reference and reuse it to align reads

$ pbmm2 index xxx.fasta xxx.mmi

B. Align contig yyy.fasta to reference genome file (xxx.mmi)

pbmm2 align xxx.mmi xxx.fasta

However, I am getting this error.

|> 20210517 18:05:07.939 -|- WARN -|- CheckPositionalArgs -|- 0x11a162dc0|| -|- Input is FASTA. Output BAM file cannot be used for polishing with GenomicConsensus!

How to fix this issue.

Thanks

assembly pbmm2 pacbio hifiasm • 237 views
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5 weeks ago
gconcepcion ▴ 80

This is just a legacy warning (pre-HiFi data) and will not prevent the command from completing.

In general, assemblies generated from HiFi data do not need to be polished.

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I have assemblies from IPA as well as from Hifiasm. However, hifiasm are longer as compared to IPA therefore, I am proceeding with hifiasm.

However, the command is being killed

zsh: killed pbmm2 align xxx.fasta yyy.fasta

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It might help to get a more detailed log from pbmm2. You can get a debug trace with --log-level DEBUG that might help you spot where/why pbmm2 is being killed.

https://github.com/PacificBiosciences/pbmm2#can-i-get-progress-output

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pbmm2 align yyy.fasta xxx.fasta --log-level DEBUG

|> 20210518 22:31:50.529 -|- INFO -|- AlignSettings -|- 0x11407cdc0|| -|- Using 8 threads for alignments. |> 20210518 22:31:50.529 -|- WARN -|- CheckPositionalArgs -|- 0x11407cdc0|| -|- Input is FASTA. Output BAM file cannot be used for polishing with GenomicConsensus! |> 20210518 22:31:50.529 -|- INFO -|- CheckPositionalArgs -|- 0x11407cdc0|| -|- READ input file: CGD-scaffold_3.fasta |> 20210518 22:31:50.529 -|- INFO -|- CheckPositionalArgs -|- 0x11407cdc0|| -|- REF input file: ctg1.fasta |> 20210518 22:31:50.530 -|- INFO -|- Index -|- 0x11407cdc0|| -|- Start reading/building index |> 20210518 22:31:51.361 -|- INFO -|- Index -|- 0x11407cdc0|| -|- Finished reading/building index |> 20210518 22:31:51.407 -|- DEBUG -|- PostInit -|- 0x11407cdc0|| -|- Minimap2 parameters based on preset: SUBREADS |> 20210518 22:31:51.407 -|- DEBUG -|- PostInit -|- 0x11407cdc0|| -|- Kmer size : 19 |> 20210518 22:31:51.407 -|- DEBUG -|- PostInit -|- 0x11407cdc0|| -|- Minimizer window size : 10 |> 20210518 22:31:51.407 -|- DEBUG -|- PostInit -|- 0x11407cdc0|| -|- Homopolymer compressed : true |> 20210518 22:31:51.407 -|- DEBUG -|- PostInit -|- 0x11407cdc0|| -|- Gap open 1 : 5 |> 20210518 22:31:51.407 -|- DEBUG -|- PostInit -|- 0x11407cdc0|| -|- Gap open 2 : 56 |> 20210518 22:31:51.407 -|- DEBUG -|- PostInit -|- 0x11407cdc0|| -|- Gap extension 1 : 4 |> 20210518 22:31:51.407 -|- DEBUG -|- PostInit -|- 0x11407cdc0|| -|- Gap extension 2 : 1 |> 20210518 22:31:51.407 -|- DEBUG -|- PostInit -|- 0x11407cdc0|| -|- Match score : 2 |> 20210518 22:31:51.407 -|- DEBUG -|- PostInit -|- 0x11407cdc0|| -|- Mismatch penalty : 5 |> 20210518 22:31:51.407 -|- DEBUG -|- PostInit -|- 0x11407cdc0|| -|- Z-drop : 400 |> 20210518 22:31:51.407 -|- DEBUG -|- PostInit -|- 0x11407cdc0|| -|- Z-drop inv : 50 |> 20210518 22:31:51.407 -|- DEBUG -|- PostInit -|- 0x11407cdc0|| -|- Bandwidth : 2000 |> 20210518 22:31:51.407 -|- DEBUG -|- PostInit -|- 0x11407cdc0|| -|- Max gap : 5000 |> 20210518 22:31:51.407 -|- DEBUG -|- PostInit -|- 0x11407cdc0|| -|- Long join flank ratio : 0.5

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