bio-long-read-sequencing-isoseq-analysis

name	bio-long-read-sequencing-isoseq-analysis
description	Discovers, classifies, filters, and quantifies full-length transcript isoforms from PacBio Iso-Seq/Kinnex (HiFi) and Oxford Nanopore (cDNA/direct-RNA) long reads, using the isoseq+pigeon pipeline, SQANTI3, and ONT tools (IsoQuant, FLAIR, Bambu, StringTie2). Covers why a novel isoform is an artifact until proven otherwise (RT template-switching, intra-priming, and 5' degradation manufacture junctions and truncations), the SQANTI3 structural categories and their trust order, the Kinnex skera-split step, orthogonal CAGE/poly-A/short-read-junction validation, and why long-read isoform quantification needs EM. Use when building a full-length isoform catalog, classifying/filtering long-read transcripts, running Iso-Seq or ONT cDNA/dRNA analysis, or judging novel-isoform reliability.
tool_type	mixed
primary_tool	SQANTI3
goal_approach_exempt	true

Version Compatibility

Reference examples tested with: isoseq 4.3+, pigeon 1.2+, SQANTI3 5.2+, pbmm2 1.13+, minimap2 2.28+, IsoQuant 3.4+.

Before using code patterns, verify installed versions match. If versions differ:

• CLI: <tool> --version then <tool> --help to confirm flags
• Python/R: pip show <pkg> / packageVersion('<pkg>') for SQANTI3/IsoQuant/Bambu

Results depend on inputs that outlive the binary version - record them:

• The reference annotation + genome version drive SQANTI3/pigeon classification; record them.
• Orthogonal support files (CAGE refTSS BED, poly-A motif/atlas, short-read STAR SJ.out) determine which novels survive; record their provenance.
• The Iso-Seq binary was renamed isoseq3 -> isoseq in v4; the classifier pigeon is a separate binary.

If code throws an error, introspect the installed tool (isoseq --help, pigeon --help, sqanti3_qc.py --help) and adapt the example to the actual API rather than retrying.

Full-Length Isoform Analysis

"Find the isoforms in my long-read RNA data" -> Build a full-length isoform catalog, then classify and filter it against the reference with orthogonal end/junction support - because discovery without curation is a catalog of artifacts.

• CLI: isoseq refine ... && isoseq collapse ... && pigeon classify ... && pigeon filter ... (PacBio), IsoQuant/FLAIR/Bambu (ONT)

The Single Most Important Modern Insight -- A Novel Isoform Is an Artifact Until Proven Otherwise

RT template-switching, intra-priming on genomic poly-A, and 5' RNA degradation actively MANUFACTURE novel junctions and truncated isoforms. So the classification + filter + orthogonal validation IS the analysis, not a QC postscript. Invert the posture from "I discovered N novel isoforms" to "I curated N novel isoforms that survived artifact filtering." Three consequences:

1. A high novel-isoform fraction is a RED FLAG, not a success - it usually means an under-powered filter or degraded RNA, not unusually rich biology.
2. ISM (incomplete-splice-match) is the RNA-degradation thermometer, not a discovery. ISMs are 5'-truncated FSMs; a high ISM fraction signals bad RNA integrity. Do not report ISMs as novel isoforms without CAGE 5' support.
3. The orthogonal validation triad is mandatory: CAGE peaks for the 5' TSS (catches 5' degradation), poly-A atlas/motif for the 3' TES (catches intra-priming), and short-read STAR junctions for splice sites (catches RT-switch/NNC junk).

SQANTI3 Structural Categories (trust order)

Reference comparison is junction-chain based. NIC > NNC in trust, always; ISM is a diagnostic, not a discovery.

Category (field value)	Meaning	Trust
FSM (full-splice_match)	every internal junction matches a reference transcript; ends may differ	highest (known); ends still need CAGE/polyA
ISM (incomplete-splice_match)	junction subset of a reference (fewer 5' exons)	low - the 5'-degradation/RT-dropoff signature; trust only with CAGE
NIC (novel_in_catalog)	novel combination of KNOWN splice sites	high among novels - RT-switching cannot fake a NIC
NNC (novel_not_in_catalog)	>=1 genuinely novel splice site	lower - where junction artifacts concentrate; needs canonical/short-read support
genic / genic_intron	overlaps introns/exons; within an intron	low - pre-mRNA / gDNA carryover
fusion	spans >=2 genes	RT-chimera until proven by short-read split reads
intergenic / antisense	no gene overlap / antisense	novel-gene candidate or artifact; needs ORF/CAGE/conservation

Mono-exon transcripts have no junctions to validate and are the false-discovery sink (intra-priming + gDNA run unchecked) - require ORF + CAGE + polyA + conservation before belief.

Platform / Tool Decision Tree

Data / goal	Tool	Why
PacBio Iso-Seq/Kinnex, turnkey	isoseq + pigeon	native PacBio collapse + SQANTI-style classify/filter, SMRT Link integrated
Any long-read transcriptome, full curation	SQANTI3	structural classification + ~50 QC descriptors + rules/ML filter + rescue; PacBio and ONT
ONT bulk discovery + quantification	IsoQuant	intron-graph; lowest novel FP rate among ONT tools
ONT, want built-in differential splicing	FLAIR	align -> correct junctions -> collapse -> diffSplice
Quantification with a precision knob	Bambu	NDR (novel discovery rate) calibrates precision; R/Bioconductor
Genome-guided assembly / hybrid short+long	StringTie2 -L (--mix)	fast long-read transcript assembly
ONT single-cell long-read isoforms	FLAMES	single-cell/spatial full-length isoforms
Differential isoform usage (DTU/DTE)	-> alternative-splicing	this skill yields the filtered set + counts and hands off

cDNA vs Direct-RNA and Spliced Alignment

PacBio Iso-Seq and ONT cDNA sequence reverse-transcribed cDNA (modifications erased; strand from primers); ONT direct-RNA sequences native RNA (true strand, poly-A length, modifications preserved, lower accuracy). Match the minimap2 preset to the chemistry:

minimap2 -ax splice ref.fa ont_cdna.fq        # ONT cDNA (orient first with pychopper)
minimap2 -ax splice -uf -k14 ref.fa drna.fq   # ONT direct RNA (stranded -> -uf, small k)
minimap2 -ax splice:hq -uf ref.fa hifi.fa     # PacBio HiFi (or pbmm2 --preset ISOSEQ)

-uf forces the forward transcript strand - correct for stranded dRNA/Iso-Seq, wrong for unoriented ONT PCR-cDNA (orient with pychopper first).

PacBio Iso-Seq / Kinnex Pipeline

# 0. Kinnex (MAS-seq) ONLY: deconcatenate the array into segmented reads FIRST
skera split movie.hifi_reads.bam mas_adapters.fasta movie.segmented.bam   # skip for classic Iso-Seq

# 1. Remove cDNA primers; 2. produce FLNC (full-length non-chimeric)
lima movie.segmented.bam primers.fasta movie.fl.bam --isoseq --peek-guess
isoseq refine movie.fl.5p--3p.bam primers.fasta movie.flnc.bam --require-polya

# 3. cluster (reference-free) or skip and align FLNC directly; 4. map; 5. collapse to isoforms
isoseq cluster2 movie.flnc.bam clustered.bam                              # cluster2 scales to large sets
pbmm2 align --preset ISOSEQ --sort ref.fa clustered.bam mapped.bam
isoseq collapse --do-not-collapse-extra-5exons mapped.bam movie.flnc.bam collapsed.gff
#   collapsed.flnc_count.txt = FLNC molecules per isoform = the real DEPTH metric

# 6. classify + filter with pigeon (needs the collapsed.sorted.gff after prepare, NOT a BAM)
pigeon prepare collapsed.gff            # sorts the transcript GFF
pigeon prepare annotation.gtf ref.fa    # sorts the annotation -> annotation.sorted.gtf, indexes genome
pigeon classify collapsed.sorted.gff annotation.sorted.gtf ref.fa \
    --fl collapsed.flnc_count.txt --cage-peak cage.refTSS.bed --poly-a polyA.motif.list
pigeon filter collapsed_classification.txt --isoforms collapsed.sorted.gff
pigeon report --exclude-singletons collapsed_classification.filtered_lite_classification.txt saturation.txt

pigeon is PacBio's productized SQANTI3 (classify/filter, NOT a quantifier). Substitute SQANTI3 itself for the full descriptor set, ML filter, rescue module, and ONT support:

sqanti3_qc.py collapsed.gff annotation.gtf ref.fa --CAGE_peak cage.bed --polyA_motif_list polyA.txt \
    --short_reads short_reads_fofn.txt    # isoforms positional defaults to GTF/GFF; add --fasta for FASTA input
sqanti3_filter.py rules collapsed_classification.txt   # or: sqanti3_filter.py ml ...

Per-Method Failure Modes

Counting ISMs as novel isoforms

Trigger: reporting incomplete-splice-match transcripts as discoveries. Mechanism: 5' RNA degradation truncates FSMs into ISMs. Symptom: inflated novel/ISM fraction tracking RNA quality, not biology. Fix: treat ISM fraction as an integrity QC; keep ISMs only with CAGE 5' support.

Intra-priming false 3' ends

Trigger: trusting 3' ends without poly-A validation. Mechanism: oligo-dT primes on a genomic internal A-stretch. Symptom: spurious short/mono-exon transcripts; perc_A_downstream_TTS >59%. Fix: SQANTI3/pigeon filter on downstream genomic A-content and poly-A motif; --require-polya alone does NOT catch this.

Believing NNC novels without scrutiny

Trigger: treating NNC like NIC. Mechanism: novel splice sites are where RT template-switching and mapping artifacts land. Symptom: novel junctions absent from short-read data. Fix: require canonical junctions or short-read SJ coverage; prefer NIC.

Feeding pigeon a BAM

Trigger: pigeon classify mapped.bam .... Mechanism: pigeon classifies the collapsed.sorted.gff after pigeon prepare, not an alignment. Symptom: wrong-input error. Fix: isoseq collapse -> pigeon prepare -> pigeon classify.

Comparing isoform counts across libraries of different depth

Trigger: raw isoform counts as abundance. Mechanism: discovery is depth-unsaturated; truncated reads are multi-isoform-compatible. Symptom: deeper libraries "have more isoforms"; double-counted abundance. Fix: rarefaction curve (--exclude-singletons); EM quantification (Bambu/IsoQuant/NanoCount), not raw FLNC counts.

Quantitative Thresholds

Threshold	Source	Rationale
perc_A_downstream_TTS > 59-60% = intra-priming	SQANTI (Tardaguila 2018)	genomic A-rich window means the poly-A was internal, not the real tail
novel junction trusted if canonical OR short-read cov >= 3	SQANTI3 rules filter	a single criterion for RT-switch/NNC artifacts
ML filter needs >= 250 Reference-Match FSM	SQANTI3	enough true-positive labels to train; else falls back to rules
exclude singletons (1-FLNC) for saturation	pigeon report	singletons are the dominant unreliable novel bucket
FLNC count = depth metric	isoseq collapse	independently sequenced full-length molecules, before clustering/dedup

Common Errors

Error / symptom	Cause	Solution
isoseq3: command not found	renamed in v4	use isoseq (subcommands unchanged)
pigeon classify wrong input	fed a BAM	give the collapsed.sorted.gff after pigeon prepare
Huge novel-isoform count	filter skipped/underpowered	run pigeon/SQANTI3 filter with CAGE/polyA/short-read support
Many mono-exon novels	intra-priming / gDNA carryover	filter on poly-A; require ORF/CAGE for mono-exon
Wrong-strand spliced alignment	-uf on unoriented cDNA	orient with pychopper, or drop -uf for cDNA
Isoform counts not comparable across samples	depth-unsaturated discovery	EM quantification + rarefaction curve

References

• Tardaguila M, de la Fuente L, Marti C, et al. 2018. SQANTI: extensive characterization of long-read transcript sequences for quality control in full-length transcriptome identification and quantification. Genome Res 28(3):396-411.
• Pardo-Palacios FJ, Arzalluz-Luque A, Kondratova L, et al. 2024. SQANTI3: curation of long-read transcriptomes for accurate identification of known and novel isoforms. Nat Methods 21(5):793-797.
• Prjibelski AD, Mikheenko A, Joglekar A, et al. 2023. Accurate isoform discovery with IsoQuant using long reads. Nat Biotechnol 41(7):915-918.
• Tang AD, Soulette CM, van Baren MJ, et al. 2020. Full-length transcript characterization of SF3B1 mutation in chronic lymphocytic leukemia (FLAIR). Nat Commun 11:1438.
• Chen Y, Sim A, Wan YK, et al. 2023. Context-aware transcript quantification from long-read RNA-seq data with Bambu. Nat Methods 20(8):1187-1195.
• Al'Khafaji AM, Smith JT, Garimella KV, et al. 2024. High-throughput RNA isoform sequencing using programmed cDNA concatenation (MAS-ISO-seq/Kinnex). Nat Biotechnol 42(4):582-586.

Related Skills

• long-read-alignment - Spliced alignment of cDNA/direct-RNA (splice/splice:hq, -uf)
• basecalling - Direct-RNA (RNA004) basecalling; cDNA vs direct-RNA chemistry
• nanopore-methylation - Direct-RNA modifications are separate from isoform structure
• alternative-splicing/long-read-splicing - Long-read splicing analysis (define the boundary)
• alternative-splicing/isoform-switching - Differential isoform usage (DTU) downstream
• alternative-splicing/differential-splicing - Differential splicing downstream
• rna-quantification/tximport-workflow - Transcript-level quantification downstream
• genome-annotation/eukaryotic-gene-prediction - Long-read isoforms as annotation evidence