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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
Barcode name rules:
- please start with an alphabetic character prefix to avoid later confusion of barcode name and index,
- dashes are not allowed,
- no duplicate names.
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 --hifi-preset SYMMETRIC
HiFi run from FASTQ with asymmetric barcodes:
lima <movie>.hifi_reads.fq.gz barcodes.fasta <movie>.demux.fastq --hifi-preset ASYMMETRIC
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