********************
INTRODUCTION
********************

Last Updated: 12/13/2023

This README file describes the contents in this directory.

This directory contains data from Kinnex 16S rRNA kit. Note that some of the datasets
were released prior to the official Kinnex kit names and may still retain an older name
such as "MAS" or "MAS16S".

To learn more about Kinnex, visit https://pacb.com/kinnex


********************
SAMPLE
********************

Refer to the samplesheet in each DATA- directory for per-sample details. All the samples
in this repository come from one or more of the following sources.

1) ZymoFecal:

ZymoBIOMICS Fecal Reference with TruMatrix™ Technology
Catalog# D6323
Lot# 195710

2) ZymoGut:

ZymoBIOMICS Gut Microbiome Standard
Catalog# D6331
Lot# 213554

3) ATCCeven:

ATCC 20 Strain Even Mix Genomic Material
Catalog# MSA-1002
Lot# 70001383


4) ATCCstagger:

ATCC 20 Strain Staggered Mix Genomic Material
Catalog# MSA-1003
Lot# 70044980


********************
METHODS
********************

Library Preparation: 
Procedure & Checklist - Preparing Kinnex libraries from 16s rRNA amplicons
https://www.pacb.com/wp-content/uploads/Procedure-checklist-Preparing-Kinnex-libraries-from-16S-rRNA-amplicons.pdf

Sequencing: 
Revio system with Revio polymerase kit and Revio sequencing plate
or
Sequel II/IIe system with Sequel II polymerase 2.2 and Sequel II sequencing kit 2.0

Run time: 
Revio – 24 hr movie
Sequel II/IIe - 24 hr movie

Analysis: 
Read segmentation & Demultiplexing in SMRT Link
GitHub https://github.com/PacificBiosciences/HiFi-16S-workflow
   
********************
FILE DESCRIPTION
********************

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 16S amplicons. segmented.bam contains S-reads that 
have the expected order of Kinnex adapters and is the file used in carrying the subsequent analyses.

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


========================
2-Demux
========================

This directory contains demultiplexed per-sample BAM and fastq files. Refer to the samplesheet.txt in 
each DATA- directory for the mapping of barcode pairs to sample names. The files in this directory 
will generally have the following file name structures.

2-Demux/
└── xxxxx.Kinnex16S_Fwd_xx--Kinnex16S_Rev_xx[.bam, .fastq.gz]


========================
3-16S analysis
========================

This directory contains the output results from running HiFi-16S-workflow (formerly named pb-16S-nf)

3-pb-16S-nf-analysis/
├── cmd.sh
├── visualize_biom.html
└── ...etc...


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

[1] skera.how https://skera.how/
[2] 16S nextflow pipeline https://github.com/PacificBiosciences/HiFi-16S-workflow
[3] kinnex https://pacb.com/kinnex


Research use only. Not for use in diagnostic procedures. 
© 2023 Pacific Biosciences of California, Inc. (“PacBio”). All rights reserved. 
Information in this document is subject to change without notice. 
PacBio assumes no responsibility for any errors or omissions in this document. 
Certain notices, terms, conditions and/or use restrictions may pertain to your 
use of PacBio products and/or third-party products. Refer to the applicable 
PacBio terms and conditions of sale and to the applicable license terms at pacb.com/license. 
Pacific Biosciences, the PacBio logo, PacBio, Circulomics, Omniome, SMRT, 
SMRTbell, Iso-Seq, Sequel, Nanobind, SBB, Revio, and Onso are trademarks of PacBio.