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Input

Reads

Reads can be in following formats:

Data type File type Example
CLR unaligned PacBio BAM <movie>.subreads.bam
CCS unaligned PacBio BAM <movie>.reads.bam
HiFi unaligned PacBio BAM <movie>.hifi_reads.bam
CLR XML <movie>.subreadset.xml
CCS / HiFi XML <movie>.consensusreadset.xml
CCS / HiFi FASTA [gzipped] <movie>.fasta[.gz]
CCS / HiFi FASTQ [gzipped] <movie>.fastq[.gz]

Barcodes

Barcodes have to be in FASTA format <barcodes>.fasta, one entry per barcode sequence, no duplicate sequences, only upper-case bases, orientation agnostic (forward or reverse-complement, but NOT reversed). Example:

>bc1000
CTCTACTTACTTACTG
>bc1001
GTCGTATCATCATGTA
>bc1002
AATATACCTATCATTA

Please name your barcodes with an alphabetic character prefix to avoid later confusion of barcode name and index. Duplicate names or sequences are not permitted.

Can I have upper- and lower-case bases in my barcodes?
You can, but lima is case-insensitive and will convert them to upper case before the alignment step.

Output

It is advised to use the same output as input file type. This compatibility matrix explains what in- and output combinations are possible.

Following additional auxilliary files are generated:

  • <prefix>.lima.summary, a human-readable summary of barcoded yield and failures
  • <prefix>.lima.report, in-depth diagnostics for each ZMW
  • <prefix>.lima.counts, ZMW counts per barcode pair and mean barcode score
  • <prefix>.lima.clips, clipped barcode regions with --dump-clips
  • <prefix>.lima.guess, barcode pairs and if they were selected with --guess / --peek-guess
  • <prefix>.removed.bam, unbarcoded ZMWs with --dump-removed

Each output .bam file is accompanied by a .bam.pbi index file.

In- and output compatibility matrix:

For CLR data, only XML and BAM are valid in- and output file types.

For CCS / HiFi data, use following compatibility matrix:

In/Out XML BAM FASTQ FASTA
XML YES YES YES YES
BAM YES YES YES YES
FASTQ no no YES YES
FASTA no no no YES

Example executions

HiFi run from BAM with symmetric barcodes:

lima <movie>.hifi_reads.bam barcodes.fasta <movie>.demux.bam --same --ccs --min-score 80

HiFi run from FASTQ with asymmetric barcodes:

lima <movie>.hifi_reads.fq.gz barcodes.fasta <movie>.demux.fastq --different --ccs --min-score 80

CLR run from XML with symmetric barcodes:

lima <movie>.subreadset.xml barcodes.fasta <movie>.demux.subreadset.xml --same

CLR run from BAM with asymmetric barcodes:

lima <movie>.subreads.bam barcodes.fasta <movie>.demux.bam --different

HiFi run from FASTA with single-sided barcodes:

lima <movie>.fasta barcodes.fasta <movie>.demux.fasta --ccs --min-score 80 --single-side

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