README  (Last Updated 10/01/2019)

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INTRODUCTION
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   This README file describes the contents in this directory.

   This dataset contains raw, intermediate, and processed files for an 
Alzheimer brain Iso-Seq dataset. The library was sequenced on the 
Sequel II system and processed using SMRTLink 8.0 followed by community 
tool analysis. For more  information on Iso-Seq® methods[1], bioinformatics 
analysis, see the PacBio Iso-Seq GitHub[2] and additional references below.


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SAMPLE
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300ng Total RNA - Alzheimer's Disease Brain Sample (BioChain lot #507294) 

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METHODS
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Library Preparation: 
Iso-Seq® Express Template Preparation for Sequel® and Sequel® II Systems 

Sequencing: 
Sequel II System with Sequel II Binding Kit 1.0 and Sequel II Sequencing Kit 1.0 (4 rxn)

Run time: 
24hr movie + 2hr pre-extension 

Analysis: 
SMRTlink 8.0 "IsoSeq" protocol, followed by mapping to hg38 reference genome 
and collapsed into  non-redundant transcript set.[3] 

Post-mapping filtering using SQANTI2 software[4].
   
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FILE DESCRIPTION
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WHAT FILES SHOULD I USE? 
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Users wishing to immediately make use the processed, mapped, 
filtered results should use the following files:

PolishedMappedTranscripts/
|---- after-SQANTI2filter
|   |---- Alzheimer_IsoSeq2019.postFilter.abundance.txt 
|   |---- Alzheimer_IsoSeq2019.postFilter.faa 
|   |---- Alzheimer_IsoSeq2019.postFilter.fasta 
|   |---- Alzheimer_IsoSeq2019.postFilter.gtf 


UCSC Genome Browser Session:
https://genome.ucsc.edu/s/Magdoll/2019_Alzheimer8M



We DO NOT recommend most users re-analyzing from raw (subreads.bam) 
or intermediate (FLNC) data.

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Raw Subreads
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The RawMovie/ folder contains the movie BAM file. 

RawMovie/
|---- m64014_190506_005857.adapters.fasta 
|---- m64014_190506_005857.sts.xml 
|---- m64014_190506_005857.subreads.bam 
|---- m64014_190506_005857.subreads.bam.pbi 
|---- md5sums.txt


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Intermediate FLNC Reads
========================

The FullLengthReads/ directory contains the full-length, non-concatemer (FLNC) 
reads in both BAM and FASTQ format. 

FullLengthReads/
|---- flnc.bam 
|---- flnc.fasta
|---- flnc.filter_summary.json 
|---- flnc.report.csv 
|---- md5sums.txt

========================
Mapped and Filtered Transcripts
========================

The PolishedMappedTranscripts/ directory contains two subfolders. 

The "before-SQANTI2filter" directory contains the results of mapping the 
Iso-Seq output (full-length, high-quality isoform sequences) to the hg38 
reference genome, then collapsing the result using Cupcake [3] scripts with 
99% coverage and 95% identity cutoff. The collapsed results are delineated 
into the two samples based on the associated FLNC read count. The SQANTI2 
results (.classification.txt, .junctions.txt, and _sqanti_report.pdf)
are also included. 

The "after-SQANTI2filter" subdirectory contains the same files, but after 
running the SQANTI2 filtering script to remove library artifacts. For more 
information on the SQANTI2 filtering, see [4].


PolishedMappedTranscripts/
|---- after-SQANTI2filter
|   |---- Alzheimer_IsoSeq2019.postFilter.abundance.txt 
|   |---- Alzheimer_IsoSeq2019.postFilter.faa 
|   |---- Alzheimer_IsoSeq2019.postFilter.fasta 
|   |---- Alzheimer_IsoSeq2019.postFilter.gtf 
|   |---- Alzheimer_IsoSeq2019.postFilter.sqanti_classification.txt 
|   |---- Alzheimer_IsoSeq2019.postFilter.sqanti_junctions.txt 
|   |---- Alzheimer_IsoSeq2019.postFilter.sqanti_report.pdf 
|   |---- md5sums.txt
|---- before-SQANTI2filter
    |---- Alzheimer_IsoSeq2019.preFilter.abundance.txt 
    |---- Alzheimer_IsoSeq2019.preFilter.faa
    |---- Alzheimer_IsoSeq2019.preFilter.fasta 
    |---- Alzheimer_IsoSeq2019.preFilter.gtf 
    |---- Alzheimer_IsoSeq2019.preFilter.sqanti_classification.txt 
    |---- Alzheimer_IsoSeq2019.preFilter.sqanti_junctions.txt 
    |---- Alzheimer_IsoSeq2019.preFilter.sqanti_report.pdf 
    |---- md5sums.txt
  
4. REFERENCES

[1] PacBio Iso-Seq Landing Page: https://www.pacb.com/applications/rna-sequencing/
[2] PacBio Iso-Seq GitHub Wiki: https://github.com/PacificBiosciences/IsoSeq_SA3nUP
[3] Community Tool Cupcake: https://github.com/Magdoll/cDNA_Cupcake
[4] Community Tool SQANTI2: https://github.com/Magdoll/SQANTI2/


For Research Use Only. Not for use in diagnostic procedures.  Copyright 2019, 
Pacific Biosciences of California, Inc. All rights reserved. The data provided in 
these files is subject to change without notice and Pacific Biosciences assumes no 
responsibility for any errors or omissions. Certain notices, terms, conditions and/or 
use restrictions may pertain to your use of Pacific Biosciences data, products and/or 
third party products. Please refer to the applicable Pacific Biosciences Terms and 
Conditions of Sale and to the applicable license terms at 
http://www.pacificbiosciences.com/licenses.html.