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INTRODUCTION
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Last Updated: 06/01/2023

This README file describes the contents in this directory.

This is a preliminary data release of MAS-Seq for Bulk Iso-Seq kit. Data is subject 
to modification until the final release.



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SAMPLE
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Revio-HG002-1
Vendor – Coriell
Lot No – n/a
HG002 cells were purchased from Coriell and grown in RPMI1640 w/Glutamax media with 
16% FBS and 0.5% Penicillin-Streptomycin. RNA was isolated from 10x10^6 HG002 cells 
using Trizol reagent and Phasemaker tubes. RNA quality was assessed using a Bioanalyzer.



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METHODS
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Library Preparation: 
(Unreleased) Procedure & Checklist - Preparing MAS-Seq libraries using MAS-Seq for bulk Iso-Seq kit

Sequencing: 
Revio system with Revio polymerase kit and Revio sequencing plate

Run time: 
Revio – 24 hr movie

Analysis: 
(Unreleased) Read segmentation and Iso-Seq workflow

   
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FILE DESCRIPTION
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Each sample will contain the following folders:


========================
1-Sreads
========================

This directory contains segmented reads that have been processed by Read segmentation (or skera) [1]
to produce S-reads that represent the original cDNA molecules. segmented.bam contains S-reads that 
have the expected order of MAS adapters and is the file used in carrying the subsequent analyses.

1-Sreads/
├── segmented.bam
└── segmented.non_passing.bam


========================
2-FLNC
========================

This directory contains full-length, non-concatemer (FLNC) reads that have been removed of the 
5' and 3' cDNA primer as well as the polyA tail. FLNC reads are oriented from 5'->3' based on 
the asymmetry of the cDNA primers.


2-FLNC/
└── flnc.bam


========================
3-ClusterMap
========================

This directory contains HQ cluster sequences by clustering FLNC reads at the isoform level de novo. 
The HQ cluster reads are then mapped using pbmm2 (minimap2 wrapper) to hg38.

3-ClusterMap
├── clustered.bam
└── mapped.bam


========================
4-Collapse
========================

This directory contains the result of collapsing redundant isoforms after HQ cluster sequences were 
mapped to the genome. Each collapsed isoform has an unique ID `PB.X.Y` with an associated FLNC read count.

4-Collapse
├── collapsed_transcripts.fasta 
├── collapsed_transcripts.flnc_count.txt 
├── collapsed_transcripts.gff 
├── collapsed_transcripts.group.txt
└── collapsed_transcripts.read_stat.txt 


========================
5-Pigeon
========================


This directory lists the the total set of unique transcripts as a result of mapping the dedup reads 
to the genome, collapsed into transcripts, classified and filtered against Gencode using pigeon. 
Read about pigeon at [2]. 

The classification.txt and junctions.txt are the output from pigeon showing the per-isoform and 
per-junction-per-isoform classification results against Gencode annotation. The GFF3 file shows 
the exonic structures of the transcript isoforms. 


5-Pigeon/
├── pigeon_classification.filtered_lite_classification.txt 
├── pigeon_classification.filtered_lite_junctions.txt 
├── pigeon.sorted.filtered_lite.gff 
└── pigeon_classification.filtered_lite_pigeon_report.pdf



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REFERENCES
********************

[1] skera.how https://skera.how/

[2] isoseq.how https://isoseq.how/




Research use only. Not for use in diagnostic procedures.
 © 2023 Pacific Biosciences of California, Inc. (“PacBio”). All rights reserved. 
The data provided in these files and the information in this document are subject to change without notice. 
PacBio assumes no responsibility for any errors or omissions in the files or this document. Certain notices, 
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