Awesome

pbmarkdup takes one or multiple sequencing chips of an amplified libray as HiFi reads and marks or removes duplicates.

Availability

Latest pbmarkdup can be installed via bioconda package pbmarkdup.

Please refer to our official pbbioconda page for information on Installation, Support, License, Copyright, and Disclaimer.

Latest Version

Version 1.1.0: Full changelog here

Execution

Input: HiFi reads from one or multiples movies in PacBio BAM (.ccs.bam), PacBio dataset (.consensusreadset.bam), file of file names (.fofn), FASTQ (optionally gzipped), or FASTA (optionally gzipped) format.

Output: HiFi reads with duplicates marked in a format inferred from the file extension: HiFi BAM (.bam); FASTQ (.fastq); FASTQ (.fasta); bgzipped FASTQ (.fastq.gz); bgzipped FASTA (.fasta.gz); or SMRT Link XML (.consensusreadset.xml) which also generates a corresponding BAM file.

Run on a single movie:

pbmarkdup movie.ccs.bam output.bam

Run on multiple movies

pbmarkdup movie1.fasta movie2.fasta output.fasta

Run on multiple movies and output duplicates in separate file

pbmarkdup movie1.ccs.bam movie2.fastq uniq.fastq --dup-file dups.fasta

FAQ

How does pbmarkdup work?

pbmarkdup employs a reference-free HiFi comparison scheme to avoid biases from alignment-based methods. Naïve implementation would perform all-vs-all alignments to determine if two reads originate from the identical molecule and are only due to PCR duplication. This would be abysmally slow. Instead, pbmarkdup relies on a hierarchical comparison workflow to rapidly detect reads that are likely from the same molecule.

1. Compare each read to all reads that are smaller in length but at most 10 bp
2. Check if their minimizers are similar
3. Perform alignment of the first and last 250 bp in both directions
4. Cluster reads and deterministically determine cluster representative

Additional heuristics are employed to enable ultra-fast comparison.

How much memory is required?

A low-memory footprint has been a primary focus of pbmarkdup. In order to enable rapid comparison of HiFi reads, all reads have to be stored in memory. As an example, a 20x human dataset would consume 60 GB memory just to store the sequences plus minimizers which can be a multiple, resulting in >200 GB memory consumption.
Alternatively, pbmarkdup only stores the first and last 250 bp of each read with 2 byte/base and aggressively thins minimizers. This results in 8 GB memory for a 60 GB dataset (unreleased version 0.3.0).

Why are input files parsed twice?

In order to keep memory footprint to a minimum, we trade reading input files twice instead of storing everything in memory. The goal was to support processing multiple movies with a standard server.

Does pbmarkdup work on my CLR data?

No. The purpose of this tool to mark PCR duplicates for amplicon HiFi libraries after CCS calling.

How are duplicates marked?

Duplicates have annotated read names in FASTA and FASTQ; FASTA example

>m64013_191117_050515/67104/ccs DUPLICATE=m64013_191117_050515/3802014/ccs DS=2

shows a marked duplicate read m64013_191117_050515/67104/ccs that is a duplicate of m64013_191117_050515/3802014/ccs and the this molecule has been sequenced 2 times. Accordingly, the read that has been selected as the representative of the molecule:

>m64013_191117_050515/3802014/ccs DS=2

In BAM format, tags ds:i: provides the number of reads of this molecule and du:Z: the name of the representative read.

Can I print summary information

Yes, use --log-level INFO and get following summary:

LIBRARY         READS    UNIQUE MOLECULES    DUPLICATE READS
----------  ----------  ------------------  -----------------
<Unnamed>        25000       24948 (99.8%)          52 (0.2%)
SS-lib             496         493 (99.4%)           3 (0.6%)
----------  ----------  ------------------  -----------------
TOTAL            25496       25441 (99.8%)          55 (0.2%)

For each library, BAM @RG library tag LB or <Unnamed> for FASTA/Q input, number and percentages of unique and duplicate reads are listed per row.

Can I mark duplicates across different libraries?

Use --cross-library to mark agnostic of library names.

Can I store duplicates in an extra file?

Use --dup-file FILE to store duplicates in an extra file. The format of this file can be different to the output file.

Can I remove duplicates?

Use --rmdup.

What input / output combinations are allowed

Input as rows, outputs as columns:

Allowed combination example:

pbmarkdup movie1.ccs.bam movie2.fastq movie3.fasta out.fasta

Forbidden combination example:

pbmarkdup movie2.fastq movie3.fasta out.fastq

Is there a progress report?

Yes. With --log-level INFO, pbmarkdup provides status to stderr.

Licenses

PacBio® tool pbmarkdup, distributed via Bioconda, is licensed under BSD-3-Clause-Clear.

Changelog

• 1.1.0:
  • SMRT Link v25.1 release
  • Use same tag syntax for FASTX as BAM
• 1.0.3:
  • Revio release for SMRT Link 12.0
• 1.0.2:
  • Official SMRT Link 10.0 release
  • Minor internal fix in memory compression
• 1.0.0:
  • Official SMRT Link 9.0 release
  • Improve memory consumption
• 0.2.0:
  • Initial release

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