--- title: "Long-Read Isoform Identification and Quantification with IsoQuant" canonical: "https://help.biobam.com/space/OED0324/3539337217/Long-Read%20Isoform%20Identification%20and%20Quantification%20with%20IsoQuant" format: markdown --- # Introduction Isoform Identification and Quantification in *[Ursus arctos horribilis](https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=116960)**.* ### Dataset Description This dataset contains two files of long reads sequenced by PacBio Sequel technology, representing adipose tissue of a male bear, once in active phase and once in hibernation phase. - Organism: *[Ursus arctos horribilis](https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=116960)**.* - Instrument: PacBio Sequel ### Publication [Tseng, E., Underwood, J. G., Evans Hutzenbiler, B. D., Trojahn, S., Kingham, B., Shevchenko, O., ... & Kelley, J. L. (2022). Long-read isoform sequencing reveals tissue-specific isoform expression between active and hibernating brown bears (Ursus arctos). ](https://academic.oup.com/g3journal/article/12/3/jkab422/6472356)*[G3](https://academic.oup.com/g3journal/article/12/3/jkab422/6472356)*[, ](https://academic.oup.com/g3journal/article/12/3/jkab422/6472356)*[12](https://academic.oup.com/g3journal/article/12/3/jkab422/6472356)*[(3), jkab422.](https://academic.oup.com/g3journal/article/12/3/jkab422/6472356)
Abstract Understanding hibernation in brown bears (Ursus arctos) can provide insight into some human diseases. During hibernation, brown bears experience periods of insulin resistance, physical inactivity, extreme bradycardia, obesity, and the absence of urine production. These states closely mimic aspects of human diseases such as type 2 diabetes, muscle atrophy, as well as renal and heart failure. The reversibility of these states from hibernation to active season enables the identification of mediators with possible therapeutic value for humans. Recent studies have identified genes and pathways that are differentially expressed between active and hibernation seasons in bears. However, little is known about the role of differential expression of gene isoforms on hibernation physiology. To identify both distinct and novel mRNA isoforms, full-length RNA-sequencing (Iso-Seq) was performed on adipose, skeletal muscle, and liver from three individual bears sampled during both active and hibernation seasons. The existing reference genome annotation was improved by combining it with the Iso-Seq data. Short-read RNA-sequencing data from six individuals were mapped to the new reference annotation to quantify differential isoform usage (DIU) between tissues and seasons. We identified differentially expressed isoforms in all three tissues, to varying degrees. Adipose had a high level of DIU with isoform switching, regardless of whether the genes were differentially expressed. Our analyses revealed that DIU, even in the absence of differential gene expression, is an important mechanism for modulating genes during hibernation. These findings demonstrate the value of isoform expression studies and will serve as the basis for deeper exploration into hibernation biology.
### Original Data - PacBio NCBI Project: [PRJNA727613](https://www.ncbi.nlm.nih.gov/bioproject/PRJNA727613) - NCBI Genome and Annotation: [Ursus arctos](https://www.ncbi.nlm.nih.gov/datasets/taxonomy/9644/) # Bioinformatic Analysis ## Isoform Definition and Quantification using IsoQuant ### Application Long-Read Isoform Definition and Quantification (IsoQuant). ### Input - PacBio Long-Reads dataset in FASTQ format: [sample 29 (active)](https://drive.google.com/file/d/1bItJQ1M90J04n-xbjH021E49TLw8V6gm/view?usp=sharing) and [sample 30 (hibernation)](https://drive.google.com/file/d/1aY9sdFhnodLQRkuhO8oQWj2z0CECKUWb/view?usp=sharing). - [NCBI genome](https://drive.google.com/file/d/1gJxrVsZGoLP_jN55Ek-CBhQmtj11oVT2/view?usp=sharing) in FASTA format. - [NCBI annotation](https://drive.google.com/file/d/1lrzKCOd_FP6FBVW-ZMQkIabnjIKvhKhr/view?usp=sharing) in .gtf format (or in .db format using gtftools) ### Parameters #### Input Read Strandness: None #### Annotation Inputs Use Reference Annotation: True Detailed Gene Database: False #### Algorithm Options Data Type: PacBio CCS or FLNC Reads Full-Length Transcripts: True Matching Strategy: Precise Splice Correction: Default PacBio Model Construction: Full-Length PacBio Report Mono-Exonic Transcripts: True Transcript Quantification: With Ambiguous Gene Quantification: With Inconsistent #### Output Output File Prefix: URSUS_1male_adipose Save Transcript Model Annotations: True Reference Gene Counts: True Reference Transcript Counts: True Transcript Counts: True ### Execution Time Converting .gtf to .db with gtftools: 50m Running IsoQuant: 32m Total: 1h 22m ### Output - [URSUS_1male_adipose.transcript_models.gtf](https://drive.google.com/file/d/1uBRDbtXmPbIAnKirOM0RJymHrcwCZMsi/view?usp=sharing): Transcriptome annotation in GTF format. You can use it as input to SQANTI3. - [ursus_1male_adipose_gene_grouped_counts.box](https://drive.google.com/file/d/1VM0mCP_2GZug_3ibdTG34rzXj-xsYeG9/view?usp=sharing): Gene-level quantification of reference genes. Can be exported to a .txt file. - [ursus_1male_adipose_reference_transcript_grouped_counts.box](https://drive.google.com/file/d/1VXE2rk4ly4jmBwDOdIlZvRelwqdeOOQq/view?usp=sharing): Transcript-level quantification of reference transcripts. Can be exported to a .txt file. - [ursus_1male_adipose_transcript_model_grouped_counts.box](https://drive.google.com/file/d/1a9ENN1PhkWVP70JP2-gVLpMvOmQ6uBT3/view?usp=sharing): Transcript-level quantification of isoforms defined by IsoQuant. Can be exported to a .txt file. - [isoquant_report.box](https://drive.google.com/file/d/1GQYe22g1sWjgYKwz-rgKUgxZZCS8Uzxe/view?usp=sharing): Summary Report - [isoforms_length.box](https://drive.google.com/file/d/1mc3IpNStqIoFzthD-IjEB2i7vMqneeTD/view?usp=sharing): Isoform Length Distribution