Bio Long Read Sequencing Isoseq Analysis

What it does

When your agent uses it

• Building a full-length isoform catalog
• Classifying/filtering long-read transcripts
• Running Iso-Seq
• ONT cDNA/dRNA analysis

Example prompts

• “/bio-long-read-sequencing-isoseq-analysis”

Requirements

• A Bash shell

Workflow steps

3 steps, taken from the first numbered list in SKILL.md.

1. 1A high novel-isoform fraction is a RED FLAG, not a success - it usually means an under-powered filter or degraded RNA, not unusually rich…
2. 2ISM (incomplete-splice-match) is the RNA-degradation thermometer, not a discovery. ISMs are 5'-truncated FSMs; a high ISM fraction signals…
3. 3The orthogonal validation triad is mandatory: CAGE peaks for the 5' TSS (catches 5' degradation), poly-A atlas/motif for the 3' TES…

What it can do on your machine

Read from SKILL.md and the folder at commit d91ed3d. It shows what the files ask for, not the result of running them.

• Tool permissions

  Pre-approves nothing: there is no allowed-tools line, so your agent's usual permission prompts apply.

  From allowed-tools in the SKILL.md frontmatter.

• Runs code

  Ships script files (Shell), which the agent can run.

  Shell commands in SKILL.md call:

  • pip

  From the folder's file list and the shell code blocks in SKILL.md.

• Network

  No URLs in SKILL.md. Its commands use pip, which can reach the network depending on how they are called.

  From URLs in SKILL.md, links to its own repository left out.

• Credentials

  Names no API keys, tokens, secrets or passwords.

  From names ending in _API_KEY, _TOKEN, _SECRET, _KEY or _PASSWORD in SKILL.md.

Context cost

Bio Long Read Sequencing Isoseq Analysis loads about 3.4k tokens when it runs. Until then it costs about 207 tokens; SKILL.md has 1,234 words of instructions outside code blocks.

Always · name and description, kept in context so the agent knows when to use it

~207

When it runs · the whole SKILL.md, loaded when a task matches

~3.4k

Estimates: characters ÷ 4, the usual rule of thumb; real counts depend on the model's tokenizer. Scripts and assets cost tokens only if the agent reads them.

Safety

Automated static check — not a guarantee. Review scripts before installing. It scans the text of SKILL.md for risky patterns (piping downloads into a shell, reading credential files, hidden Unicode, destructive commands); files beside SKILL.md are not scanned.

SKILL.md

The full file from GPTomics/bioSkills at commit d91ed3d, republished under its MIT licence (© GPTomics). 1,234 words, ~3,367 tokens.

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

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)

# 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

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 ...

© GPTomics, MIT. Rendered from Markdown: HTML in the file is shown as text, images as links, and headings moved down two levels. Raw file

Files

SKILL.md and 3 other files in long-read-sequencing/isoseq-analysis of GPTomics/bioSkills.

• SKILL.md
• examples/isoseq_workflow.sh
• examples/sqanti3_qc.sh
• usage-guide.md

Open the folder on GitHubat commit d91ed3d

Used in 1 other repository

We found 1 copy of this SKILL.md (exact, near-identical or edited) in other folders, from 1 other GitHub owner. This page covers the copy in GPTomics/bioSkills, which our catalogue first saw on October 7, 2026.

• PKU-YuanGroup/OpenAI4Sskills/bioskills/bio-long-read-sequencing-isoseq-analysis