Long-Read Sequencing Guide

What Is Long-Read Sequencing?

Standard WGS (from Illumina, Novogene, Dante Labs, etc.) produces short reads — millions of DNA fragments typically 150 base pairs long. Two companies offer a fundamentally different approach: reading much longer stretches of DNA in a single pass.

Oxford Nanopore Technology (ONT)

ONT sequencers (MinION, PromethION, GridION) push a strand of DNA through a tiny protein pore. As each nucleotide passes through, it changes the electrical current. The device reads these current changes in real time to determine the sequence.

• Read lengths: 1,000 to 100,000+ bp (some reads exceed 1 million bp)
• Accuracy: ~99% with R10.4.1 chemistry and super-accuracy basecalling (Q20+)
• Common data format: FASTQ (basecalled from FAST5/POD5 raw signal)
• Vendors/services: ONT direct (MinION, PromethION), some clinical labs

PacBio HiFi (High-Fidelity)

PacBio sequencers (Sequel II, Revio) use a different approach: a polymerase enzyme copies a circular DNA template while a camera records fluorescent signals from each incorporated nucleotide. By reading the same circle multiple times, errors cancel out.

• Read lengths: 10,000-25,000 bp
• Accuracy: >99.9% (Q30+, comparable to Illumina)
• Common data format: Unaligned BAM or FASTQ (HiFi/CCS reads)
• Vendors/services: PacBio direct, Revio service providers, some clinical labs

Why Use Long Reads?

Long reads solve problems that short reads cannot:

Capability	Short Reads (150bp)	Long Reads (10-100Kbp)
SNPs and small indels	Excellent	Excellent (HiFi) / Good (ONT)
Structural variants (>50bp)	Limited	Excellent
Repeat expansions	Indirect (ExpansionHunter)	Direct measurement
Phasing (which allele is on which chromosome)	Statistical	Read-based, much more accurate
Segmental duplications / tandem repeats	Mostly invisible	Resolvable
Methylation	Requires bisulfite conversion	Native (ONT)

---

Pipeline Compatibility

What the long-read branch covers: alignment (script 2b), small variants with Clair3 (3e) and structural variants with Sniffles2 (4c). After that, the VCF-based steps below work on the result. Nothing else in this pipeline is long-read specific: no repeat genotyping, no paralog or CYP2D6 resolution, no methylation and no phasing. For those, use the vendor workflows, which run the long-read tools named on this page: PacBio's HiFi-human-WGS-WDL and Oxford Nanopore's wf-human-variation.

Not every step in this pipeline works with long-read data. Here is the full breakdown.

Works As-Is (No Changes Needed)

These steps take a VCF or BAM and work identically regardless of read technology:

Step	Tool	Notes
5	AnnotSV	Takes any SV VCF — point it at Sniffles2 output
6	ClinVar Screen	VCF-only, technology-independent
7	PharmCAT	VCF-only — requires VCF + .tbi symlinked to vcf/ (see below)
11	ROH Analysis	VCF-only via plink2
12	Mito Haplogroup	VCF-only via haplogrep3
13	VEP Annotation	VCF-only
14	Imputation Prep	VCF-only
17	CPSR	VCF-only
22	SURVIVOR Merge	Takes any SV VCF set
23	Clinical Filter	VCF-only
24	HTML Report	Aggregates existing outputs
25	PRS	VCF-only via pgsc_calc
26	Ancestry	VCF-only via pgsc_calc (with the ancestry panel)
27	CPIC Lookup	Reads PharmCAT JSON output

Needs Long-Read Specific Scripts

Step	Short-Read Tool	Long-Read Alternative	Script
2	minimap2 (-x sr)	minimap2 (-x map-ont / -x map-hifi)	scripts/02b-alignment-longread.sh
3	DeepVariant (WGS model)	Clair3 or DeepVariant (ONT/PACBIO model)	scripts/03e-clair3.sh
4	Manta	Sniffles2	scripts/04c-sniffles2.sh

Does NOT Work with Long Reads

These tools are specifically designed for short-read data and will produce incorrect results or crash with long-read input:

Step	Tool	Why It Fails	Alternative
1b	fastp	Expects paired-end Illumina reads; no long-read mode	NanoPlot, LongQC, or chopper for long-read QC
2a	BWA-MEM2	Short-read aligner only; cannot handle reads >500bp	Use minimap2 with long-read preset
3a	GATK HaplotypeCaller	Active-region model assumes short reads; poor with long reads	Clair3 or DeepVariant long-read mode
3b	FreeBayes	Bayesian model tuned for short reads	Clair3 or DeepVariant long-read mode
3c	Strelka2	Scoring model trained on BWA-MEM short-read data	Clair3 or DeepVariant long-read mode
3d	Octopus	Haplotype model designed for short reads	Clair3 or DeepVariant long-read mode
4	Manta	Illumina-specific insert size model	Sniffles2
4a	TIDDIT	Short-read coverage/discordance model	Sniffles2
4b	GRIDSS	Assembly-based, short-read specific	Sniffles2
9	ExpansionHunter	Illumina short-read graph model; expects paired-end data	TRGT (PacBio) or Straglr (ONT), outside this pipeline (vendor workflows)
15	duphold	Re-genotypes SVs using short-read depth models	Not needed — Sniffles2 QUAL scores are reliable
18	CNVpytor	Read-depth model calibrated for short reads	Sniffles2 detects CNVs natively
19	Delly	Paired-end and split-read model	Sniffles2
21	Cyrius (CYP2D6)	Short-read depth-based star allele caller	Paraphase (long-read CYP2D6 resolver), outside this pipeline (vendor workflows)

---

Step-by-Step: Long-Read Analysis

Prerequisites

Same reference data as the short-read pipeline:

export GENOME_DIR=/path/to/your/data
# Reference genome and index should already exist from initial setup
# See docs/00-reference-setup.md

Step 1: Prepare Your Data

Place your long-read data in the sample directory:

# Create sample directory
mkdir -p ${GENOME_DIR}/your_sample/fastq/

# Copy or symlink your long-read FASTQ
# ONT: typically one FASTQ per run (basecalled from FAST5/POD5)
cp /path/to/ont_reads.fastq.gz ${GENOME_DIR}/your_sample/fastq/your_sample.fastq.gz

# PacBio: typically one FASTQ or unaligned BAM per cell
cp /path/to/hifi_reads.fastq.gz ${GENOME_DIR}/your_sample/fastq/your_sample.fastq.gz
# OR for unaligned BAM:
cp /path/to/hifi_reads.bam ${GENOME_DIR}/your_sample/fastq/your_sample.bam

Note: long-read data is single-end (no R1/R2 pairs). The alignment script expects a single file, not paired files.

Step 2: Align Reads

# For Oxford Nanopore
PLATFORM=ont ./scripts/02b-alignment-longread.sh your_sample

# For PacBio HiFi
PLATFORM=hifi ./scripts/02b-alignment-longread.sh your_sample

Output: ${GENOME_DIR}/your_sample/aligned_longread/your_sample_sorted.bam

The script auto-detects input files in this order: 1. fastq/your_sample.fastq.gz 2. fastq/your_sample_lr.fastq.gz 3. fastq/your_sample.fq.gz 4. fastq/your_sample.bam (unaligned BAM)

Override with: INPUT=${GENOME_DIR}/your_sample/fastq/custom_reads.fastq.gz PLATFORM=ont ./scripts/02b-alignment-longread.sh your_sample

Note: The INPUT path must be physically inside GENOME_DIR because Docker only mounts that directory. Symlinks that point outside GENOME_DIR will not work — Docker cannot follow them. Copy or move the file instead.

Step 3: Variant Calling (SNPs + Indels)

You have two options:

Option A: Clair3 (recommended for long reads)

# For ONT
PLATFORM=ont ./scripts/03e-clair3.sh your_sample

# For PacBio HiFi
PLATFORM=hifi ./scripts/03e-clair3.sh your_sample

Output: ${GENOME_DIR}/your_sample/vcf_clair3/your_sample.vcf.gz

Clair3 uses deep-learning models trained specifically on long-read error profiles. It auto-selects the correct model based on your PLATFORM setting.

Option B: DeepVariant (long-read mode)

DeepVariant 1.10.0+ also supports long reads with dedicated models:

source versions.env   # from the repository root
REF_FASTA=reference/GRCh38_no_alt_analysis_set.fasta   # see 00-reference-setup.md#the-reference-path-on-every-page
SAMPLE=your_sample

docker run --rm \
  --cpus 8 --memory 32g \
  -v "${GENOME_DIR}:/genome" \
  "${DEEPVARIANT_IMAGE}" \
  /opt/deepvariant/bin/run_deepvariant \
    --model_type=ONT_R104 \
    --ref="/genome/${REF_FASTA}" \
    --reads="/genome/${SAMPLE}/aligned_longread/${SAMPLE}_sorted.bam" \
    --output_vcf="/genome/${SAMPLE}/vcf/${SAMPLE}.vcf.gz" \
    --num_shards=8

# For PacBio, use --model_type=PACBIO instead of ONT_R104

Clair3 vs DeepVariant for long reads:

Aspect	Clair3	DeepVariant
ONT accuracy	Excellent (purpose-built)	Good (newer models improving)
PacBio HiFi accuracy	Excellent	Excellent
Speed	Generally faster	Slower (more compute-heavy)
Indel calling	Very good	Very good
Community support	Active (HKU)	Active (Google)
Model updates	Frequent (matches new chemistries)	Periodic

For PacBio HiFi data, both tools perform comparably. For ONT data, Clair3 tends to have an edge because its models are updated more frequently to match new ONT basecalling improvements.

Step 4: Structural Variant Calling

# Default: uses aligned_longread/ directory
./scripts/04c-sniffles2.sh your_sample

# Or explicitly:
ALIGN_DIR=aligned_longread ./scripts/04c-sniffles2.sh your_sample

Output: ${GENOME_DIR}/your_sample/sv_sniffles/your_sample_sv.vcf.gz

Sniffles2 excels at detecting all SV types from long reads. Typical output for a 30X long-read genome: - 15,000-25,000 SVs (vs 7,000-9,000 from short-read Manta) - Much better at insertions (short reads systematically miss these) - Better breakpoint resolution (often single-nucleotide) - Reliable in repetitive regions where short reads struggle

Step 5: Continue with Standard Pipeline Steps

After alignment and variant calling, most downstream steps work normally. Point them at the long-read outputs:

SAMPLE=your_sample

# ClinVar screen (uses VCF from Clair3)
VCF_DIR=vcf_clair3 ./scripts/06-clinvar-screen.sh "$SAMPLE"

# PharmCAT (uses VCF from Clair3)
# PharmCAT needs the VCF + .tbi index at the default vcf/ location.
# Symlink or copy both files:
mkdir -p "${GENOME_DIR}/${SAMPLE}/vcf"
ln -sf "../vcf_clair3/${SAMPLE}.vcf.gz" "${GENOME_DIR}/${SAMPLE}/vcf/${SAMPLE}.vcf.gz"
ln -sf "../vcf_clair3/${SAMPLE}.vcf.gz.tbi" "${GENOME_DIR}/${SAMPLE}/vcf/${SAMPLE}.vcf.gz.tbi"
./scripts/07-pharmacogenomics.sh "$SAMPLE"

# VEP annotation (uses VCF from Clair3)
VCF_DIR=vcf_clair3 ./scripts/13-vep-annotation.sh "$SAMPLE"

# Annotate Sniffles2 SVs
SV_VCF="${GENOME_DIR}/${SAMPLE}/sv_sniffles/${SAMPLE}_sv.vcf.gz" ./scripts/05-annotsv.sh "$SAMPLE"

---

Docker Images Used

Tool	Image	Size
minimap2	MINIMAP2_IMAGE	~30 MB
samtools	SAMTOOLS_IMAGE	~200 MB
Clair3	CLAIR3_IMAGE	~3 GB (includes all models)
Sniffles2	SNIFFLES_IMAGE	~200 MB
DeepVariant	DEEPVARIANT_IMAGE	~5 GB

Pinned in versions.env; Image versions lists the current tag.

Pre-pull images before your first run:

source versions.env   # from the repository root
docker pull "${MINIMAP2_IMAGE}"
docker pull "${SAMTOOLS_IMAGE}"
docker pull "${CLAIR3_IMAGE}"
docker pull "${SNIFFLES_IMAGE}"

---

ONT-Specific Notes

Basecalling Matters

ONT raw data (FAST5/POD5) must be basecalled before entering this pipeline. Use the latest Dorado basecaller with the super accuracy (SUP) model for best variant calling:

# This happens BEFORE the pipeline — on a GPU machine
dorado basecaller sup pod5_dir/ > reads.bam
dorado demux reads.bam  # if barcoded

The Clair3 model (r1041_e82_400bps_sup_v500) assumes R10.4.1 chemistry with SUP basecalling. Using a different chemistry or basecaller may reduce accuracy.

Read Length Distribution

ONT generates a wide range of read lengths. Very short reads (<1kb) add noise. If your N50 is below 5kb, consider filtering:

No step of this pipeline filters reads. If you want to drop reads under 1 kb, run a read filter such as chopper on the FASTQ yourself before script 2b.

Methylation

ONT natively detects methylation (5mC, 6mA) during basecalling with Dorado. Methylation tags are stored in the BAM as MM/ML tags. This pipeline does not process methylation data. When the input is an unaligned BAM, script 2b carries the MM/ML tags into the aligned BAM, where the vendor workflows above can read them.

---

PacBio-Specific Notes

HiFi vs CLR

This pipeline supports HiFi (CCS) reads only, not older CLR (continuous long reads). HiFi reads are generated by PacBio Sequel II, Sequel IIe, and Revio systems using circular consensus sequencing.

If you received CLR data, you'll need to generate CCS reads first using ccs or pbccs (PacBio tool, outside this pipeline).

Kinetic Tags

PacBio HiFi BAMs may contain kinetic modification tags (fi, fp, ri, rp) for methylation detection. Like ONT methylation, these are preserved in alignment but not processed by this pipeline.

---

Hybrid Analysis (Short + Long Reads)

If you have both short-read and long-read data for the same sample, you can run both pipelines and combine results:

1. Short-read pipeline: standard 02-alignment.sh + 03-deepvariant.sh + 04-manta.sh
2. Long-read pipeline: 02b-alignment-longread.sh + 03e-clair3.sh + 04c-sniffles2.sh
3. Merge variant calls:
4. SNPs/indels: use benchmark-variants.sh to compare, then take the intersection for high-confidence calls
5. SVs: use 22-survivor-merge.sh with both Manta and Sniffles2 VCFs for consensus SVs

This is the gold standard approach when both data types are available.

---

Troubleshooting

"minimap2 runs out of memory"

Long-read alignment uses more memory than short-read because the index is loaded into RAM. Increase Docker memory limit:

# In the script, change --memory 16g to --memory 24g or higher

"Clair3 model not found"

The Docker image CLAIR3_IMAGE bundles models at /opt/models/. If you get a model-not-found error:

1. Verify the image is pulled: docker images | grep clair3
2. Check available models: docker run --rm "${CLAIR3_IMAGE}" ls /opt/models/ (after source versions.env in the repository root)
3. If your ONT chemistry is different (e.g., R9.4.1), look for a matching model in the container

"Sniffles2 produces 0 SVs"

• Verify the BAM is from long-read alignment (not short-read): samtools stats your.bam | grep "average length"
• Average read length should be >1000bp for ONT or >5000bp for HiFi
• Check that the BAM is sorted and indexed

"DeepVariant long-read model fails"

• Use --model_type=ONT_R104 for ONT (not ONT alone)
• Use --model_type=PACBIO for PacBio HiFi
• DeepVariant 1.10.0 requires the exact model type string — check the DeepVariant release notes for your version

"VCF downstream steps fail on Clair3 output"

Steps 6 (ClinVar), 13 (VEP), and 17 (CPSR) accept a VCF_DIR env var:

VCF_DIR=vcf_clair3 ./scripts/06-clinvar-screen.sh your_sample
VCF_DIR=vcf_clair3 ./scripts/13-vep-annotation.sh your_sample

For steps that don't support VCF_DIR (e.g., PharmCAT), symlink the Clair3 VCF:

ln -s ../vcf_clair3/${SAMPLE}.vcf.gz ${GENOME_DIR}/${SAMPLE}/vcf/${SAMPLE}.vcf.gz
ln -s ../vcf_clair3/${SAMPLE}.vcf.gz.tbi ${GENOME_DIR}/${SAMPLE}/vcf/${SAMPLE}.vcf.gz.tbi

---

Expected Output Comparison

Rough numbers for a 30X human genome:

Metric	Short-Read (Illumina)	ONT (R10.4.1 SUP)	PacBio HiFi (Revio)
SNPs	~4.5M	~4.3M	~4.5M
Indels	~1.0M	~0.8M	~1.0M
PASS rate	~93%	~85-90%	~93%
SVs (total)	~7-9K (Manta)	~20-25K (Sniffles2)	~18-22K (Sniffles2)
SV insertions	~500 (poor)	~8-10K (excellent)	~7-9K (excellent)
Alignment time	1-2 hours	1-3 hours	1-2 hours
Variant calling time	2-4 hours (DV)	2-4 hours (Clair3)	2-4 hours (Clair3)

The biggest advantage of long reads is SV detection — especially insertions, which short reads systematically miss.