Convert raw Nanopore signal data (FAST5/POD5) to nucleotide sequences using Dorado basecaller. Covers model selection, GPU acceleration, modified base detection, and quality filtering. Use when processing raw Nanopore data before alignment. Note: Guppy is deprecated; use Dorado for all new analyses.
npxskills add majiayu000/claude-skill-registry--skill bio-basecallingLoading…
Convert raw electrical signal from Nanopore sequencing into nucleotide sequences.
Dorado is ONT's current production basecaller, replacing Guppy. It offers better accuracy and speed.
dorado basecaller sup pod5_dir/ > calls.bam
dorado basecaller fast pod5_dir/ > calls.bam
dorado basecaller hac pod5_dir/ > calls.bam
dorado basecaller sup pod5_dir/ > calls.bam
| Model | Speed | Accuracy | Use Case |
|---|---|---|---|
| fast | Fastest | Lower | Quick preview |
| hac | Medium | High | General use |
| sup | Slowest | Highest | Publication quality |
dorado download --model [email protected]
dorado basecaller [email protected] pod5_dir/ > calls.bam
dorado download --list
dorado basecaller sup pod5_dir/ --emit-fastq > calls.fastq
dorado basecaller sup,5mCG_5hmCG pod5_dir/ > calls_mods.bam
dorado basecaller sup,5mCG pod5_dir/ > calls_5mc.bam
dorado basecaller sup,6mA pod5_dir/ > calls_6ma.bam
dorado basecaller sup pod5_dir/ --device cuda:0 > calls.bam
dorado basecaller sup pod5_dir/ --device cuda:0,1 > calls.bam
dorado basecaller sup pod5_dir/ --device cpu > calls.bam
dorado basecaller sup pod5_dir/ --batchsize 64 > calls.bam
dorado duplex sup pod5_dir/ > duplex.bam
dorado basecaller sup pod5_dir/ --kit-name SQK-NBD114-24 > calls.bam
dorado demux calls.bam --output-dir demuxed/ --kit-name SQK-NBD114-24
dorado basecaller sup pod5_dir/ --trim adapters > calls.bam
dorado basecaller sup pod5_dir/ --no-trim > calls_untrimmed.bam
dorado basecaller sup pod5_dir/ --resume-from calls.bam > calls_complete.bam
Guppy is deprecated and no longer receiving updates. Use Dorado for all new analyses. Guppy examples below are only for maintaining legacy pipelines.
guppy_basecaller \
-i fast5_dir/ \
-s output_dir/ \
-c dna_r10.4.1_e8.2_400bps_sup.cfg \
--device cuda:0
guppy_basecaller \
-i fast5_dir/ \
-s output_dir/ \
-c dna_r10.4.1_e8.2_400bps_fast.cfg \
--num_callers 8 \
--cpu_threads_per_caller 4
guppy_basecaller \
-i fast5_dir/ \
-s output_dir/ \
-c dna_r10.4.1_e8.2_400bps_hac.cfg \
--device cuda:0
guppy_basecaller \
-i fast5_dir/ \
-s output_dir/ \
-c dna_r10.4.1_e8.2_400bps_sup.cfg \
--device cuda:0
guppy_basecaller --print_workflows
ls /opt/ont/guppy/data/*.cfg
guppy_basecaller \
-i fast5_dir/ \
-s output_dir/ \
-c dna_r10.4.1_e8.2_400bps_modbases_5mc_cg_sup.cfg \
--device cuda:0
guppy_basecaller \
-i fast5_dir/ \
-s output_dir/ \
-c dna_r10.4.1_e8.2_400bps_sup.cfg \
--device cuda:0 \
--barcode_kits SQK-NBD114-24
guppy_basecaller \
-i fast5_dir/ \
-s output_dir/ \
-c dna_r10.4.1_e8.2_400bps_sup.cfg \
--device cuda:0 \
--bam_out \
--index
POD5 is the new format replacing FAST5.
pod5 convert fast5 fast5_dir/*.fast5 --output pod5_dir/
pod5 merge pod5_dir/*.pod5 --output merged.pod5
pod5 inspect reads input.pod5
pod5 inspect summary input.pod5
pod5 subset input.pod5 --output subset.pod5 --read-id-file read_ids.txt
gunzip -c calls.fastq.gz | chopper -q 10 -l 500 | gzip > filtered.fastq.gz
gunzip -c calls.fastq.gz | \
awk 'BEGIN{OFS="\n"} {h=$0; getline seq; getline plus; getline qual;
split(h, a, " "); split(a[4], q, "=");
if(q[2] >= 10) print h, seq, plus, qual}' | \
gzip > q10_filtered.fastq.gz
gunzip -c calls.fastq.gz | NanoFilt -q 10 -l 500 | gzip > filtered.fastq.gz
NanoPlot --fastq calls.fastq.gz -o qc_report/ --plots hex dot
NanoPlot --bam calls.bam -o qc_report/
pycoQC -f sequencing_summary.txt -o pycoqc_report.html
seqkit stats calls.fastq.gz
awk 'NR%4==2 {sum+=length($0); count++} END {print "Reads:", count, "Mean length:", sum/count}' calls.fastq
| Model | Use |
|---|---|
| dna_r10.4.1_e8.2_400bps_fast | Quick analysis |
| dna_r10.4.1_e8.2_400bps_hac | Routine work |
| dna_r10.4.1_e8.2_400bps_sup | High accuracy |
| Model | Use |
|---|---|
| dna_r9.4.1_450bps_fast | Quick analysis |
| dna_r9.4.1_450bps_hac | Routine work |
| dna_r9.4.1_450bps_sup | High accuracy |
#!/bin/bash
INPUT=$1
OUTPUT=$2
MODEL=${3:-sup}
mkdir -p $OUTPUT
if [ -d "$INPUT/fast5" ]; then
echo "Converting FAST5 to POD5..."
pod5 convert fast5 $INPUT/fast5/*.fast5 --output $OUTPUT/pod5/
INPUT_DIR="$OUTPUT/pod5"
else
INPUT_DIR="$INPUT"
fi
echo "Basecalling with $MODEL model..."
dorado basecaller $MODEL $INPUT_DIR > $OUTPUT/calls.bam
echo "Converting to FASTQ..."
samtools fastq $OUTPUT/calls.bam | gzip > $OUTPUT/calls.fastq.gz
echo "Filtering..."
gunzip -c $OUTPUT/calls.fastq.gz | chopper -q 10 -l 500 | gzip > $OUTPUT/filtered.fastq.gz
echo "QC report..."
NanoPlot --fastq $OUTPUT/filtered.fastq.gz -o $OUTPUT/qc/
echo "Done!"
| Model | VRAM Required | Speed (R10.4.1) |
|---|---|---|
| fast | 4 GB | ~450 bases/s |
| hac | 8 GB | ~200 bases/s |
| sup | 12 GB | ~50 bases/s |
dorado basecaller sup pod5_dir/ --batchsize 32 > calls.bam
dorado basecaller fast pod5_dir/ --device cpu > calls.bam
nvidia-smi -l 1
watch -n 1 nvidia-smi
Create or update AgentSkills. Use when designing, structuring, or packaging skills with scripts, references, and assets.
Create or update AgentSkills. Use when designing, structuring, or packaging skills with scripts, references, and assets.
Set up and use 1Password CLI (op). Use when installing the CLI, enabling desktop app integration, signing in (single or multi-account), or reading/injecting/running secrets via op.
CLI to manage emails via IMAP/SMTP. Use `himalaya` to list, read, write, reply, forward, search, and organize emails from the terminal. Supports multiple accounts and message composition with MML (MIME Meta Language).
Create or update AgentSkills. Use when designing, structuring, or packaging skills with scripts, references, and assets.
Create or update AgentSkills. Use when designing, structuring, or packaging skills with scripts, references, and assets.
Set up and use 1Password CLI (op). Use when installing the CLI, enabling desktop app integration, signing in (single or multi-account), or reading/injecting/running secrets via op.
CLI to manage emails via IMAP/SMTP. Use `himalaya` to list, read, write, reply, forward, search, and organize emails from the terminal. Supports multiple accounts and message composition with MML (MIME Meta Language).