long-read-splicing

Name: long-read-splicing
Author: GPTomics

1.1kstars185forks

How to install long-read-splicing?

gptomics/bioskills/long-read-splicing

$npx -y skills add GPTomics/bioSkills --skill long-read-splicing

Installs into the current project.

Summary

Analyzes alternative splicing from PacBio Iso-Seq (HiFi, Kinnex/MAS-Iso-seq) and Oxford Nanopore (direct cDNA, direct RNA, R10.4.1+) long-read RNA-seq with full-isoform resolution. Tools include FLAIR (correct/collapse/quantify/diffSplice for PacBio + ONT), IsoQuant (de-novo or a

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SKILL.md

1	## Version Compatibility
2
3	Reference examples tested with: FLAIR 2.0+, IsoQuant 3.5+, Bambu 3.4+, SQANTI3 5.4+, minimap2 2.26+, samtools 1.19+, rMATS-long 0.2+, IsoSeq3 4.0+
4
5	Before using code patterns, verify installed versions match. If versions differ:
6	- Python: `pip show <package>` then `help(module.function)` to check signatures
7	- R: `packageVersion('<pkg>')` then `?function_name` to verify parameters
8	- CLI: `<tool> --version` then `<tool> --help` to confirm flags
9
10	If code throws ImportError, AttributeError, or TypeError, introspect the installed
11	package and adapt the example to match the actual API rather than retrying.
12
13	# Long-Read Splicing Analysis
14
15	Full-length long-read sequencing solves problems that short-read AS cannot: anchor-length-limited microexon detection, complex multi-exon isoform deconvolution, recursive splicing in long introns, and transcript-quantification uncertainty in DTU. The 2024-2026 transition: long-read is becoming the splicing default for high-resolution analysis.
16
17	## When Long-Read Wins
18
19	\| Question \| Why long-read wins \|
20	\|----------\|---------------------\|
21	\| Microexon detection (3-27 nt) \| Reads span the microexon entirely; no aligner anchor problem \|
22	\| Long-intron recursive splicing \| Can detect ratchet point usage (Sibley 2015 Nature) \|
23	\| Complex isoform deconvolution (TTN, MAPT, NEFM) \| Single read per isoform avoids EM ambiguity \|
24	\| DTU without quantification uncertainty \| Transcript identity is read-level, not inferred \|
25	\| Novel transcript discovery \| No annotation dependence \|
26	\| Phasing splicing with SNVs \| Allele-resolved isoforms \|
27	\| Single-cell full-length isoforms \| MAS-Iso-seq + 10X 5' is the practical SOTA \|
28	\| Cryptic splicing in TDP-43 ALS \| Full-length reads confirm cryptic exon inclusion in target transcripts \|
29
30	## Platform Selection Matrix
31
32	\| Platform \| Throughput \| Accuracy (modal) \| Best for \| Fails when \|
33	\|----------\|------------\|------------------\|----------\|------------\|
34	\| PacBio Revio HiFi (Iso-Seq) \| ~25M reads / SMRT cell \| Q30+ (CCS) \| Bulk transcript discovery; gold standard \| Cost prohibitive for very large cohorts \|
35	\| PacBio Kinnex / MAS-Iso-seq \| ~16x Iso-Seq via concatemer \| Q30+ \| High-throughput single-cell long-read \| Kinnex de-array (skera) is an extra step \|
36	\| ONT direct cDNA (R10.4.1, PCS-114) \| Millions / flowcell \| ~98% simplex, ~99% duplex \| Cost-effective; throughput \| Minor higher error than HiFi \|
37	\| ONT direct RNA (RNA004, 2024+) \| ~30M reads \| ~96-98% \| Native modifications (m6A, pseudo-U); no RT bias \| Lower throughput; higher input \|
38	\| ONT pre-R10 (R9.4.1) \| Same as R10 \| ~85-90% \| Legacy data \| Pre-R10 not recommended for splicing analysis (false novel junctions) \|
39
40	Read length: PacBio HiFi cdna typically 1-10 kb; ONT cdna 0.5-50+ kb (long-tailed). Both span typical mammalian transcripts. Direct RNA on ONT preserves true 5'/3' termini and modifications.
41
42	## Decision Tree by Use Case
43
44	\| Use case \| Recommended tools \|
45	\|----------\|--------------------\|
46	\| Bulk Iso-Seq transcript discovery in well-annotated organism \| minimap2 -ax splice:hq -> IsoQuant or Bambu -> SQANTI3 \|
47	\| Bulk ONT cDNA in well-annotated organism \| minimap2 -ax splice -uf -k14 -> IsoQuant or FLAIR -> SQANTI3 \|
48	\| End-to-end pipeline for differential analysis \| FLAIR (correct -> collapse -> quantify -> diffSplice) \|
49	\| Joint discovery + quantification with calibrated novel rate \| Bambu in R \|
50	\| De novo discovery for non-model organism \| IsoQuant with --genedb omitted \|
51	\| Event-level differential splicing on long reads \| rMATS-long \|
52	\| DTU on long-read transcript counts \| DRIMSeq -> DEXSeq/satuRn -> stageR (no Salmon Gibbs needed) \|
53	\| Hybrid short+long for cohort \| StringTie2 hybrid + FLAIR / IsoQuant \|
54	\| Single-cell full-length isoforms \| MAS-Iso-seq + 10X 5' -> FLAMES or scNanoGPS \|
55	\| Cryptic exon validation in ALS \| minimap2 -> FLAIR collapse -> manual inspection of UNC13A, STMN2 \|
56	\| ASO design with full-isoform context \| minimap2 -> IsoQuant -> SQANTI

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long-read-splicing

1.1kstars185forks

View on GitHub

How to install long-read-splicing?

gptomics/bioskills/long-read-splicing

$npx -y skills add GPTomics/bioSkills --skill long-read-splicing

Installs into the current project.

Summary

Files · 1

View on GitHub

SKILL.md

1	## Version Compatibility
2
3	Reference examples tested with: FLAIR 2.0+, IsoQuant 3.5+, Bambu 3.4+, SQANTI3 5.4+, minimap2 2.26+, samtools 1.19+, rMATS-long 0.2+, IsoSeq3 4.0+
4
5	Before using code patterns, verify installed versions match. If versions differ:
6	- Python: `pip show <package>` then `help(module.function)` to check signatures
7	- R: `packageVersion('<pkg>')` then `?function_name` to verify parameters
8	- CLI: `<tool> --version` then `<tool> --help` to confirm flags
9
10	If code throws ImportError, AttributeError, or TypeError, introspect the installed
11	package and adapt the example to match the actual API rather than retrying.
12
13	# Long-Read Splicing Analysis
14
15	Full-length long-read sequencing solves problems that short-read AS cannot: anchor-length-limited microexon detection, complex multi-exon isoform deconvolution, recursive splicing in long introns, and transcript-quantification uncertainty in DTU. The 2024-2026 transition: long-read is becoming the splicing default for high-resolution analysis.
16
17	## When Long-Read Wins
18
19	\| Question \| Why long-read wins \|
20	\|----------\|---------------------\|
21	\| Microexon detection (3-27 nt) \| Reads span the microexon entirely; no aligner anchor problem \|
22	\| Long-intron recursive splicing \| Can detect ratchet point usage (Sibley 2015 Nature) \|
23	\| Complex isoform deconvolution (TTN, MAPT, NEFM) \| Single read per isoform avoids EM ambiguity \|
24	\| DTU without quantification uncertainty \| Transcript identity is read-level, not inferred \|
25	\| Novel transcript discovery \| No annotation dependence \|
26	\| Phasing splicing with SNVs \| Allele-resolved isoforms \|
27	\| Single-cell full-length isoforms \| MAS-Iso-seq + 10X 5' is the practical SOTA \|
28	\| Cryptic splicing in TDP-43 ALS \| Full-length reads confirm cryptic exon inclusion in target transcripts \|
29
30	## Platform Selection Matrix
31
32	\| Platform \| Throughput \| Accuracy (modal) \| Best for \| Fails when \|
33	\|----------\|------------\|------------------\|----------\|------------\|
34	\| PacBio Revio HiFi (Iso-Seq) \| ~25M reads / SMRT cell \| Q30+ (CCS) \| Bulk transcript discovery; gold standard \| Cost prohibitive for very large cohorts \|
35	\| PacBio Kinnex / MAS-Iso-seq \| ~16x Iso-Seq via concatemer \| Q30+ \| High-throughput single-cell long-read \| Kinnex de-array (skera) is an extra step \|
36	\| ONT direct cDNA (R10.4.1, PCS-114) \| Millions / flowcell \| ~98% simplex, ~99% duplex \| Cost-effective; throughput \| Minor higher error than HiFi \|
37	\| ONT direct RNA (RNA004, 2024+) \| ~30M reads \| ~96-98% \| Native modifications (m6A, pseudo-U); no RT bias \| Lower throughput; higher input \|
38	\| ONT pre-R10 (R9.4.1) \| Same as R10 \| ~85-90% \| Legacy data \| Pre-R10 not recommended for splicing analysis (false novel junctions) \|
39
40	Read length: PacBio HiFi cdna typically 1-10 kb; ONT cdna 0.5-50+ kb (long-tailed). Both span typical mammalian transcripts. Direct RNA on ONT preserves true 5'/3' termini and modifications.
41
42	## Decision Tree by Use Case
43
44	\| Use case \| Recommended tools \|
45	\|----------\|--------------------\|
46	\| Bulk Iso-Seq transcript discovery in well-annotated organism \| minimap2 -ax splice:hq -> IsoQuant or Bambu -> SQANTI3 \|
47	\| Bulk ONT cDNA in well-annotated organism \| minimap2 -ax splice -uf -k14 -> IsoQuant or FLAIR -> SQANTI3 \|
48	\| End-to-end pipeline for differential analysis \| FLAIR (correct -> collapse -> quantify -> diffSplice) \|
49	\| Joint discovery + quantification with calibrated novel rate \| Bambu in R \|
50	\| De novo discovery for non-model organism \| IsoQuant with --genedb omitted \|
51	\| Event-level differential splicing on long reads \| rMATS-long \|
52	\| DTU on long-read transcript counts \| DRIMSeq -> DEXSeq/satuRn -> stageR (no Salmon Gibbs needed) \|
53	\| Hybrid short+long for cohort \| StringTie2 hybrid + FLAIR / IsoQuant \|
54	\| Single-cell full-length isoforms \| MAS-Iso-seq + 10X 5' -> FLAMES or scNanoGPS \|
55	\| Cryptic exon validation in ALS \| minimap2 -> FLAIR collapse -> manual inspection of UNC13A, STMN2 \|
56	\| ASO design with full-isoform context \| minimap2 -> IsoQuant -> SQANTI

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