Bio Basecalling

Basic Basecalling

dorado basecaller sup pod5_dir/ > calls.bam

Choose Model

dorado basecaller fast pod5_dir/ > calls.bam
dorado basecaller hac pod5_dir/ > calls.bam
dorado basecaller sup pod5_dir/ > calls.bam

Model Speed vs Accuracy

Specific Model Version

dorado download --model [email protected]
dorado basecaller [email protected] pod5_dir/ > calls.bam

List Available Models

dorado download --list

Output FASTQ Instead of BAM

dorado basecaller sup pod5_dir/ --emit-fastq > calls.fastq

Modified Base Detection

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

GPU Selection

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

Batch Size for Memory

dorado basecaller sup pod5_dir/ --batchsize 64 > calls.bam

Duplex Calling

dorado duplex sup pod5_dir/ > duplex.bam

Demultiplexing During Basecalling

dorado basecaller sup pod5_dir/ --kit-name SQK-NBD114-24 > calls.bam
dorado demux calls.bam --output-dir demuxed/ --kit-name SQK-NBD114-24

Trim Adapters

dorado basecaller sup pod5_dir/ --trim adapters > calls.bam
dorado basecaller sup pod5_dir/ --no-trim > calls_untrimmed.bam

Resume Interrupted Run

dorado basecaller sup pod5_dir/ --resume-from calls.bam > calls_complete.bam

Guppy (Deprecated - Legacy Only)

Guppy is deprecated and no longer receiving updates. Use Dorado for all new analyses. Guppy examples below are only for maintaining legacy pipelines.

Basic Basecalling

guppy_basecaller \
    -i fast5_dir/ \
    -s output_dir/ \
    -c dna_r10.4.1_e8.2_400bps_sup.cfg \
    --device cuda:0

CPU Mode

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

High Accuracy Model

guppy_basecaller \
    -i fast5_dir/ \
    -s output_dir/ \
    -c dna_r10.4.1_e8.2_400bps_hac.cfg \
    --device cuda:0

Super Accuracy Model

guppy_basecaller \
    -i fast5_dir/ \
    -s output_dir/ \
    -c dna_r10.4.1_e8.2_400bps_sup.cfg \
    --device cuda:0

List Available Configs

guppy_basecaller --print_workflows
ls /opt/ont/guppy/data/*.cfg

Modified Base Calling

guppy_basecaller \
    -i fast5_dir/ \
    -s output_dir/ \
    -c dna_r10.4.1_e8.2_400bps_modbases_5mc_cg_sup.cfg \
    --device cuda:0

Barcoding During Basecalling

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

Output BAM

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 File Handling

POD5 is the new format replacing FAST5.

Convert FAST5 to POD5

pod5 convert fast5 fast5_dir/*.fast5 --output pod5_dir/

Merge POD5 Files

pod5 merge pod5_dir/*.pod5 --output merged.pod5

Inspect POD5

pod5 inspect reads input.pod5
pod5 inspect summary input.pod5

Subset POD5

pod5 subset input.pod5 --output subset.pod5 --read-id-file read_ids.txt

Quality Filtering

Filter with Chopper (After Basecalling)

gunzip -c calls.fastq.gz | chopper -q 10 -l 500 | gzip > filtered.fastq.gz

Filter by Quality Score

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

NanoFilt (Alternative)

gunzip -c calls.fastq.gz | NanoFilt -q 10 -l 500 | gzip > filtered.fastq.gz

Basecalling QC

NanoPlot

NanoPlot --fastq calls.fastq.gz -o qc_report/ --plots hex dot
NanoPlot --bam calls.bam -o qc_report/

pycoQC (From Sequencing Summary)

pycoQC -f sequencing_summary.txt -o pycoqc_report.html

Basic Stats

seqkit stats calls.fastq.gz

awk 'NR%4==2 {sum+=length($0); count++} END {print "Reads:", count, "Mean length:", sum/count}' calls.fastq

Model Selection Guide

R10.4.1 Chemistry (Current)

R9.4.1 Chemistry (Legacy)

Complete Pipeline

Goal: Run the full Nanopore basecalling pipeline from raw signal data through quality-filtered reads with a QC report.

Approach: Convert FAST5 to POD5 if needed, basecall with Dorado, convert to FASTQ, filter with chopper, and generate NanoPlot QC.

#!/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!"

GPU Requirements

Troubleshooting

Out of Memory

dorado basecaller sup pod5_dir/ --batchsize 32 > calls.bam

Slow CPU Basecalling

dorado basecaller fast pod5_dir/ --device cpu > calls.bam

Check GPU Usage

nvidia-smi -l 1
watch -n 1 nvidia-smi

Related Skills

• long-read-alignment - Align basecalled reads
• long-read-qc - QC after basecalling
• medaka-polishing - Polish using basecalled reads
• structural-variants - SV detection from long reads