README  (Last Updated 07/22/2022)

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INTRODUCTION
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This README file describes the contents in this directory.

This dataset contains raw, reprocessed, and aligned files for multiple AAV samples.

The library was sequenced on the Sequel® II System and processed using SMRT® Link v10.1 or SMRT Link v11.0 [1], followed by community tool analysis [2].

To learn more about PacBio's gene therapy research tools offering, see: https://www.pacb.com/gene-therapy/


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SAMPLE
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2021-scAAV/rh10-CBA-eGFP 
Vendor – Vector Biolabs
Lot No – 210621-210723

2022-pAV-CMV-GFP
Serotype - AAV9
Vendor – Vigene Biosciences
Catalog No – CV10009 
Lot No (Date) - 2021.02.25

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METHODS
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DNA extraction – PureLink Viral DNA/RNA Mini Kit

Library Preparation: 

Procedure & Checklist - Preparing multiplexed AAV SMRTbell® libraries using SMRTbell prep kit 3.0

Sequencing: 

2021-scAAV-CBA-eGFP
Sequel II system with Sequel II binding kit 2.1 and Sequel II sequencing kit 2.0 (4 rxn), Sequencing primer v4

2022-ssAAV-pAV-CMF-GFP
Sequel II system with Sequel II binding kit 2.0 and Sequel II sequencing kit 2.0 (4 rxn), Sequencing primer v4

2022-ssAAV-scAAV-mix
1 part of ssAAV library mixed with 10 parts of scAAV library
Sequel II system with Sequel II binding kit 3.1 and Sequel II sequencing kit 2.0 (4 rxn), Sequencing primer v4

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

Analysis: 

Detailed commands for each step can be found [here on the AAV wiki.]
(https://github.com/Magdoll/AAV/wiki/Tutorial:-Analyzing-AAV-Data)

   
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FILE DESCRIPTION
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Each sample will contain the following folders:

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0-reads
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CCS reads after running "AAV" mode in Run Design via SL 11.0

0-reads/
|---- <movie>.hifi_reads.bam
|---- <movie>.hifi_reads.bam.pbi



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1-(demuxed)-and-mapped
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If samples are multiplexed, they are shown as demux individual samples here.
For each sample, the HiFi reads are mapped to the reference plasmid.
The key files are shown below.

1-mapped/
|---- <sample>_AAV_report.pdf
|---- <sample>.nonmatch_stat.csv.gz
|---- <sample>.per_read.csv
|---- <sample>.summary.csv
|---- <sample>.tagged.sorted.bam
|---- <sample>.tagged.sorted.bam.bai


This aligned sorted BAM file is what you can visualize using IGV.




4. REFERENCES

[1] Procedure & checklist: Preparing multiplexed AAV SMRTbell® libraries using SMRTbell prep kit 3.0
https://www.pacb.com/wp-content/uploads/Procedure-checklist-Preparing-multiplexed-AAV-SMRTbell-libraries-using-SMRTbell-prep-kit-3.0.pdf

[2] AAV GitHub Wiki: https://github.com/Magdoll/AAV


For Research Use Only. Not for use in diagnostic procedures.  Copyright 2022, 
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 
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http://www.pacificbiosciences.com/licenses.html.