f5c-v1.5

Changes from previous version:

• --m6anet option for directly generating output for m6anet as requested by @yuukiiwa
• improved log output for skipped reads due to basecalls of split reads
• update slow5lib
• Minor Bug fixes:
  • fix wrong insertion at the beginning of ss string for RNA in resquiggle (#156)
  • fix rare assertion trigged for rna004 (#177 and #163)

f5c-v1.4

Changes from v1.3 are:

• shift and scale added to sam output (sc and sh tags). See here
• methylation compare and plotting scripts added compare_methylation.py and plot_methylation.R
• update slow5lib to latest
• inbuilt rna004 support (added inbuilt rna004 9-mer model invoked via option --pore rna004, autodetected for S/BLOW5]

f5c-v1.3

Changes/new features from f5c-v1.3-beta are:

1. adding a draft 5-mer pore model for upcoming RNA004 chemistry. To use this model:

   First download the model file

   wget https://raw.githubusercontent.com/hasindu2008/f5c/v1.3/test/rna004-models/rna004.nucleotide.5mer.model
   

   Then execute event align with --kmer-model pointing to the path to the downloaded k-mer model as follows:

   f5c eventalign --rna -b reads.bam -r reads.fastq -g transciptome.fa -o eventalign.tsv --kmer-model /path/to/rna004.nucleotide.5mer.model  # --slow5 
   reads.blow5  if using S/BLOW5 input
   

   Thanks @GoekeLab for help with this model generation and testing.

2. Fixing a bug that affected the last line of SAM output being truncated sometimes (--sam in eventalign).

---

Compared f5c-v1.2, this f5c-v1.3 version (introduced in f5c-v1.3-beta) adds a new feature to f5c eventalign to write the output in PAF and SAM formats with signal-to-reference alignment information embedded as tags. This output is much more compact than the default TSV output, yet is sufficient for reconstructing the alignment (Note: As of this version signal samples corresponding to insertion are yet collapsed to the previous base. This will be improved in future versions). These SAM and PAF formats with signal alignment tags are the heart of the pileup view being implemented in Squigualiser.

• eventalign PAF output which is explained here (-c option)
• default eventalign SAM output changed (--sam option). The new SAM format is explained here. To revert to the old SAM format (same format as when Nanopolish --sam is provided), please provide --sam-out-version 1. The reason for the change in the SAM format is to support the upcoming pileup view in Squigualiser. See the screenshots below.
• -a shorthand option for --sam

DNA R10.4.1 pileup:

RNA R9.4.1 pileup:

f5c-v1.3-beta

This beta version adds a new feature to f5c eventalign to write the output in PAF and SAM formats with signal-to-reference alignment information embedded as tags. This output is much more compact than the default TSV output, yet is sufficient for reconstructing the alignment. These SAM and PAF formats with signal alignment tags are the heart of the pileup view being implemented in Squigualiser.

• eventalign PAF output which is explained here (-c option)
• default eventalign SAM output changed (--sam option). The new SAM format is explained here. To revert to the old SAM format (same format as when Nanopolish --sam is provided), please provide --sam-out-version 1. The reason for the change in the SAM format is to support the upcoming pileup view in Squigualiser. See the screenshots below.
• -a shorthand option for --sam

DNA R10.4.1 pileup:

RNA R9.4.1 pileup:

f5c-v1.2

No changes from v1.2-beta. Changes from v1.1 are as follows:

Major changes

• support for R10.4.1 flowcells (specify: --pore r10 option if FAST5, austodetected if S/BLOW5) for eventalign, call-methylation and resquiggle modules. See below for a benchmark.

Minor improvements

• sprintf_append dynamic (Sasha Jenner) #120
• prints stats on skipped reads due to low mapq, secondary mappings, etc. and warns when most reads are skipped due to lower mapq #122
• warn when more than half the reads fail to align/qc/calibration
• methylation models are unnecessarily not loaded for eventalign and resquiggle
• f4c3aaf: minor summary change
• 527ffc3: fix rsq

R10.4.1 f5c methylation calling benchmark

Correlation between whole genome NA12878 f5c CpG methylation calls (inhouse generated R10.4.1 PromethION data) and publicly available NA12878 bisulphite (no coverage filtering, that is, 1X also included):

Correlation between whole genome NA24385 f5c CpG methylation calls (inhouse generated R10.4.1 PromethION data) and publicly available NA24385 bisulphite data without any coverage filtering (no coverage filtering, that is, 1X also included):

The log-likelihood ratio for methylated vs unmethylated calls:

How R10.4.1 model training was done is detailed here. Anyone who have access to better training data may follow this tutorial to further improve the accuracy.

f5c-v1.2-beta

Major changes from 1.1 are:

• support for R10.4.1 flowcells (specify: --pore r10 option) for eventalign, call-methylation and resquigglemodules

Minor improvements:

• sprintf_append dynamic (Sasha Jenner) #120

• prints stats on skipped reads due to low mapq, secondary mappings, etc. and warns when most reads are skipped due to lower mapq #122

• warn when more than half the reads fail to align/qc/calibration

• methylation models are unnecessarily not loaded for eventalign and resquiggle

• f4c3aaf: minor summary change (Hasindu Gamaarachchi)

• 527ffc3: fix rsq (Hasindu Gamaarachchi)

f5c-v1.1

• the experimental resquiggle module (contributed by @hiruna72) for signal to basecall alignment of DNA and RNA. Output format is documented here. Usage:

  Usage: f5c [OPTIONS] reads.fastq signals.blow5
  
  options:
     -t INT                     number of processing threads [8]
     -K INT                     batch size (max number of reads loaded at once) [512]
     -B FLOAT[K/M/G]            max number of bases loaded at once [5.0M]
     -h                         help
     -o FILE                    output to file [stdout]
     -x STR                     parameter profile to be used for better performance (always applied before other options)
                                e.g., laptop, desktop, hpc; see https://f5c.page.link/profiles for the full list
     -c                         print in paf format
     --verbose INT              verbosity level [0]
     --version                  print version
     --kmer-model FILE          custom nucleotide k-mer model file (format similar to test/r9-models/r9.4_450bps.nucleotide.6mer.template.model)
     --rna                      the dataset is direct RNA
  

  

• f5c can be now built without HDF5 support (./configure --disable-hdf5 && make) for anyone who wants to easily compile and work only with S/BLOW5 files

• new option --skip-slow-idx added to f5c index:, which disable f5c from building the .idx for the slow5 file (useful when a slow5 index is already available)

• error messages improved for slow5 index

f5c-v1.0

bump version to v1.0 as the interface has been stable for adequate time

Changes from v0.9:

• updates to readme and information messages

f5c-v0.9

• minor bug fixes that only affects usability
• update slow5lib to enjoy fast indexing
• added instructions for mac m1 compilation

f5c-v0.8

• update slow5lib to 0.3.0 and introduce support for zstd and svb compressed BLOW5
• fix a minor bug (#94)
• a new option --min-recalib-events that exposes the minimum number of events to recalibrate (still experimental)
• a new option --collapse-events that collapses events that stays on the same reference k-mer (see #95)