Primrose

Predict 5mC in PacBio HiFi reads

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Primrose predicts 5-Methylcytosine (5mC) of each CpG in PacBio HiFi reads, using a Convolutional Neural Network. Methylation is assumed to be symmetric between strands. The output is reported in the forward direction with respect to the HiFi read sequence.

Availability

Latest version can be installed via bioconda package primrose.

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

Latest Version

Version 1.3.0: Full changelog here

Input Data

Input for primrose are PacBio HiFi reads with kinetics. You can generate HiFi with kinetics on the command-line, more info on ccs.how:

ccs movie.subreads.bam movie.hifi_reads.bam --hifi-kinetics

Alternatively, you can use SMRT Link on your HPC or define it directly in Run Design for SQIIe instruments.

Execution

Running primrose is as simple as:

primrose movie.hifi_reads.bam movie.5mc.hifi_reads.bam

Output Data

The output is adhering to the SAM tag specification from 9. Dec 2021, using Mm and Ml tags. It's also described in the PacBio BAM file formats as

Tag	Type	Description
MM	Z	Base modifications / methylation
ML	B,C	Base modification probabilities

Notes for ML: The continuous probability range of 0.0 to 1.0 is remapped to the discrete integers 0 to 255 inclusively. The probability range corresponding to an integer N is N/256 to (N + 1)/256.

Run Time

primrose scales nearly linear in the number of threads, achieving 2 GBases HiFi per minute on 16 cores. Memory footprint is very low with ~20 MB per thread.

$ primrose movie.hifi_reads.bam out.bam -j 16 --log-level INFO
Reads      : 100000
Yield      : 1.8 GBases
Throughput : 2.0 GBases/min
Run Time   : 54s 904ms
CPU Time   : 16m 52s
Peak RSS   : 0.313 GB

Training datasets

HiFi reads and subreads for true negative and true positive CpG methylation sites are available at https://downloads.pacbcloud.com/public/Sequel-II-CpG-training/.

The true negatives are from HG002 Whole Genome Amplification (WGA). The true positives are from HG002 WGA + CpG Methyltransferase (M.Sssl).

Changelog

• 1.3.0

  • Latest developer version

• 1.2.0

  • Included in upcoming SMRT Link version
  • Use official basemod tags MM and ML per SAM spec Feb 2022

• 1.1.0

  • Add CLI call into new @PG header lines
  • Allow multiple BAM files via XML input

• 1.0.0

  • Initial release