NucFlag

Generates nucleotide frequency plots and genome misassembly BED files. Fork of NucFreq.

Quickstart

# Requires Python>=3.11
pip install nucflag==1.0.0a4

Note

NucFlag v1.0.0-alpha.4 is not installable via bioconda at the moment.

Align long-reads to assembly.

# Align long reads to assembly generated from those reads.
minimap2 -x lr:hqae -I 8G asm.fa.gz reads.fq.gz | samtools view -bh -o asm.bam
samtools index asm.bam

Detect misassemblies.

# Call putative misassemblies from read alignments on a whole genome.
nucflag call -i asm.bam -f asm.fa.gz -o misassemblies.bed
# Or on a set of regions...
nucflag call -i asm.bam -f asm.fa.gz -b regions.bed -o misassemblies.bed
# Also runs on ONT read alignments.
nucflag call -i asm_ont_r9.bam -f asm.fa.gz -x ont_r9 -o misassemblies.bed
nucflag call -i asm_ont_r10.bam -f asm.fa.gz -x ont_r10 -o misassemblies.bed
# Provide a configfile for finer control.
nucflag call -i asm.bam -f asm.fa.gz -o misassemblies.bed -c config.toml

Visualize misassemblies in a number of ways.

# Generate NucFreq plots.
nucflag call -i asm.bam -f asm.fa.gz -d plots
# And add any number of tracks...
nucflag call -i asm.bam -f asm.fa.gz -d plots --tracks repeatmasker.bed segdups.bed
# Or generate bigWigs of specific signals and then merge them with `bigtools`.
# For use in IGV or other genome browsers.
nucflag call -i asm.bam -f asm.fa.gz -d plots \
    --output_pileup_dir bigwigs \
    --add_pileup_data cov mismatch mapq
bigwigmerge -l <(find bigwigs -name "*_first.bw") merged_first.bw

Generate status BED or breakdown showing distribution of assembly issues.

nucflag status -i misassemblies.bed > status.bed
# Or plot by "length"
nucflag breakdown -i misassemblies.bed -o breakdown -t percent

Estimate QV from BED file.

nucflag qv -i misassemblies.bed > qv.bed

Generate ideogram.

nucflag ideogram -i misassemblies.bed -o ideogram
# Add cytobands with a BED file with chrom, start, end, name, and btype.
nucflag ideogram -i misassemblies.bed -c cytobands.bed -o ideogram

Get consensus misassembly calls by intersection.

nucflag consensus -i nucflag_ont.bed nucflag_hifi.bed hmm_flagger_hifi.bed hmm_flagger_ont.bed > consensus.bed

Generate sample config.

nucflag config -x ont_r10 > config_r10.toml

Input

• BAM file of PacBio HiFi, ONT R9, or ONT R10 reads aligned to an assembly.
• (Recommended) Assembly.
• (Optional) BED file of regions.

Output

• BED file of misassemblies.
• (Optional) Plots with coverage and mismatch pileup with misassemblies flagged.
• (Optional) bigWigs of pileup signals.
• (Optional) BED file of assembly status.

Documentation

Read the docs at the NucFlag wiki for more information.