Isopedia is a scalable tool for analyzing hundreds to thousands of long-read transcriptome datasets using a read-level indexing approach.

It provides two key capabilities:

• Population-level transcript quantification and frequency profiling.
• Isoform diversity exploration, including fusion and splice-junction analysis.

• Quick Start
• How It Works
• Download Pre-built Index
• Build Your Own Index
• How to Use Isopedia
  • Isoform Query
  • Fusion Query and Discovery
  • Splice Query and Visualization
• Command Parameters
• How to Install Isopedia

• Computational Resource Usage
• Contact
• FAQ

Isopedia has two binaries:

• isopedia: main workflows (query + index building)
• isopedia-tools: helper utilities

# install isopedia from conda
conda install -c bioconda isopedia

# clone the repo and enter toy example directory
git clone https://github.com/zhengxinchang/isopedia && cd isopedia/toy_ex/

# query transcripts
isopedia isoform -i index/ -g gencode.v47.basic.chr22.gtf -o out.profile.tsv.gz # 3364 records returned

# query one splice junction
isopedia splice -i index/ -s 22:17744013,22:17750104 -o out.splice.tsv.gz # 13 records returned

# visualize splice query output
isopedia-splice-viz.py -i out.splice.tsv.gz -g gencode.v47.basic.chr22.gtf -o isopedia-splice-view

# query one fusion event (two breakpoints)
isopedia fusion -i index/ -p chr1:181130,chr1:201853853 -o ./out.fusion.tsv.gz # 0 recored returned, as hg002 is a healthy individual

# query multiple fusion events
isopedia fusion -i index/ -P fusion_query.tsv -o ./out.fusion.tsv.gz # 0 recored returned, as hg002 is a healthy individual

Typical workflow:

1. isopedia profile: extract isoform signals per sample.
2. isopedia merge: aggregate all profiles into one merged archive.
3. isopedia index: build tree index for fast query.
4. Query with isopedia isoform, isopedia fusion, or isopedia splice.

How Isopedia determines a positive hit for a query transcript

Isopedia provides pre-built indexes from public long-read RNA-seq datasets.

Checksum file:

• ftp://hgsc-sftp1.hgsc.bcm.tmc.edu//rt38520/isopedia_index_hs_v1.0.tar.xz.md5sum

Unpack:

tar -xvf isopedia_index_hs_v1.0.tar.xz

Prerequisites:

1. Long-read alignments (.bam/.cram)
2. A manifest TSV with at least two columns: sample name and profile path.

Example manifest:

name	path	platform
HG002_pb_chr22	/path/to/hg002_pb_chr22.isoform.gz	PacBio
HG002_ont_chr22	/path/to/hg002_ont_chr22.isoform.gz	ONT

Workflow:

# 1) profile each sample
isopedia profile -i ./chr22.pb.grch38.bam -o ./hg002_pb_chr22.isoform.gz
isopedia profile -i ./chr22.ont.grch38.bam -o ./hg002_ont_chr22.isoform.gz

# 2) merge profiles
ulimit -n 65535 # increase the maximum number of open file descriptors, in case of merging many samples.
isopedia merge -i manifest.tsv -o index/

# 3) build tree index
isopedia index -i index/ -m manifest.tsv

# 4) test query
isopedia isoform -i index/ -g gencode.v47.basic.chr22.gtf -o out.isoform.tsv.gz

Purpose:

• Annotate transcripts from an input GTF against the index.

Example:

# input GTF must be sorted
gffread -T -o- origin.gtf | sort -k1,1 -k4,4n | gffread - -o query.sorted.gtf

isopedia isoform -i index/ -g query.sorted.gtf -o isoform.out.tsv.gz

Current FORMAT string in output header:

• CPM:COUNT:FSM_CPM:FSM_COUNT:EM_CPM:EM_COUNT:INFO

Per-sample values include abundance/count components for total, FSM, and EM estimates.

Please note that CPM values are calculated by normalizing across all input transcript queries, as Isopedia expects the input GTF to represent a complete transcriptome. If you query only a subset of transcripts, the CPM values will not be meaningful.

fusion supports three modes:

1. Breakpoint query (--pos or --pos-bed)
2. Gene-region discovery (--gene-gtf)
3. Region-pair detailed read output (--region)

Examples:

# single breakpoint pair
isopedia fusion -i index/ -p chr1:1000,chr2:2000 -o fusion.single.tsv.gz

# batch breakpoint pairs from bed-like file
isopedia fusion -i index/ -P fusion_breakpoints.bed -o fusion.batch.tsv.gz

# discover candidate fusions from gene GTF
isopedia fusion -i index/ -G gene.gtf -o fusion.discovery.tsv.gz

# detailed read-level output for two regions
isopedia fusion -i index/ -r chr8:92017266-92017466,chr21:34859374-34859574 -o fusion.region.tsv.gz

Breakpoint-query output columns:

• chr1, pos1, chr2, pos2, id, min_read, positive/sample_size, left_isoforms, right_isoforms, then per-sample counts.

Gene-discovery output columns:

• geneA_name, geneB_name, total_evidences, total_samples, is_two_strand, AtoB_primary_start, AtoB_primary_end, AtoB_supp_start, AtoB_supp_end, BtoA_primary_start, BtoA_primary_end, BtoA_supp_start, BtoA_supp_end, then per-sample counts.

Region-pair detailed output columns:

• chr1, start1, end1, chr2, start2, end2, main_exon_count1, supp_segment_count2, query_part, main_isoforms, supp_aln_regions, sample_name.

Purpose:

• Query isoforms overlapping a splice junction and optionally visualize them.

Examples:

# single splice query
isopedia splice -i index/ -s chr22:41100500,chr22:41101500 -o splice.out.tsv.gz

# batch splice query
isopedia splice -i index/ -S splice_queries.bed -o splice.batch.tsv.gz

# visualize
python script/isopedia-splice-viz.py \
  -i splice.out.tsv.gz \
  -g gencode.v47.basic.annotation.gtf \
  -t script/isopedia-splice-viz-temp.html \
  -o isopedia-splice-view

splice output columns:

• id, chr1, pos1, chr2, pos2, total_evidence, cpm, matched_sj_idx, dist_to_matched_sj, n_exons, start_pos_left, start_pos_right, end_pos_left, end_pos_right, splice_junctions, then per-sample values.

splice per-sample FORMAT:

• COUNT:CPM:START|END|STRAND

Parameter lists below are based on the current CLI in source.

Core options:

• -i, --idxdir <IDXDIR>
• -g, --gtf <GTF>
• -o, --output <OUTPUT>
• -f, --flank <FLANK> (default: 10)
• -m, --min-read <MIN_READ> (default: 1)
• --info
• -n, --num-threads <NUM_THREADS> (default: 4)
• EM options: --em-max-iter, --em-conv-min-diff, --em-chunk-size, --em-effective-len-coef, --em-damping-factor, --min-em-abundance
• TSS/TES options: --no-check-tss-tes, --tss-degrad-bp, --tes-degrad-bp, --terminal-tolerance-bp
• Cache options: -c, --cached-nodes, --cached-chunk-num, --cached-chunk-size-mb
• --output-tmp-shard-counts
• --verbose

Core options:

• -i, --idxdir <IDXDIR>
• Query selectors: -p, --pos, -P, --pos-bed, -r, --region, -G, --gene-gtf
• -o, --output <OUTPUT>
• -f, --flank <FLANK> (default: 10)
• -m, --min-read <MIN_READ> (default: 1)
• Cache options: -c, --cached_nodes, --cached-chunk-number, --cached-chunk-size-mb
• --verbose

Core options:

• -i, --idxdir <IDXDIR>
• Query selectors: -s, --splice or -S, --splice-bed
• -o, --output <OUTPUT>
• -f, --flank <FLANK> (default: 10)
• -m, --min-read <MIN_READ> (default: 1)
• Cache options: -c, --cached_nodes, --cached-chunk-number, --cached-chunk-size-mb
• --verbose

Core options:

• Input selectors: -i, --bam or -g, --gtf
• -r, --reference <REFERENCE> for CRAM
• -o, --output <OUTPUT>
• --mapq <MAPQ> (default: 5)
• --use-secondary, --rname, --tid, --gid, --verbose

Core options:

• -i, --input <INPUT>
• -o, --outdir <OUTDIR>
• -c, --chunk-size <CHUNK_SIZE> (default: 1000000)

Core options:

• -i, --idxdir <IDXDIR>
• -m, --manifest <MANIFEST>
• -t, --threads <THREADS> (default: 4)

Core options:

• -l, --list
• -n, --name <NAME>
• -u, --url <URL>
• -m, --manifest <MANIFEST>
• -o, --outdir <OUTDIR> (default: .)

conda install -c zhengxinchang isopedia

• https://github.com/zhengxinchang/isopedia/releases

isopedia-<version>.linux.tar.gz is compiled on Amazon Linux 2 (GCC 7.3, glibc 2.26, binutils 2.29.1). If your distribution is not compatible, use isopedia-<version>.musl.tar.gz.

Rust and Cargo are required.

git clone https://github.com/zhengxinchang/isopedia.git
cd isopedia
cargo build --release
cargo build --release --target x86_64-unknown-linux-musl

Isopedia 1.4.0 benchmark on 1,007 long-read transcriptome datasets from SRA and ENCODE.

• Hardware: AMD Ryzen 9 7940HX, 64 GB RAM.

Q: Can Isopedia be used in a "transcript discovery" mode — for example, to retrieve all transcripts indexed for a given gene or within a genomic region, without providing a GTF file in advance?

A: Isopedia is designed as a genotyper rather than a caller. It always requires a GTF file as input, meaning users need to specify the transcript structures they want to query. Gene-level or coordinate-range discovery queries (e.g., "retrieve all transcripts for gene N" or "all transcripts between coordinates A–B") fall under the scope of a transcript caller, which is outside Isopedia's current functionality.