Wiki » History » Revision 5
Revision 4 (Katie Lennard, 06/26/2019 03:23 PM) → Revision 5/10 (Katie Lennard, 06/26/2019 03:25 PM)
# Wiki
# Sample processing details from Pieter de Waal
'The Illumina® 16S metagenomics workflow will be used to analyze the three differently sourced milk types. Each sample will be analyzed in duplicate, producing a total number of six extracted libraries. A positive control will also be included in the analyses. The hypervariable V3 and V4 regions of the 16S ribosomal RNA gene (16S rRNA) will be amplified, using an Illumina® pre-designed primer pair. Barcoding for multiplexing of the samples will entail the use of the Nextera XT®-Index Kit.'
From Jeanne Korsman (Jeanne.Korsman@cpgr.org.za):
"16S Amplicon PCR Forward Primer = 5' TCGTCGGCAGCGTC _AGATGTGTATAAGAGACAG_ CCTACGGGNGGCWGCAG TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG
(Equivalent to the 16S "337F" primer)
16S Amplicon PCR Reverse Primer = 5' GTCTCGTGGGCTCGG _AGATGTGTATAAGAGACAG_ GACTACHVGGGTATCTAATCC GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC
(Equivalent to the 16S "800R" primer)
The underlined bases are the sequencing primer binding site, so these should not be present in any of the sequences. The sequences on the 5'end are where the barcodes are attached. They are also available in the 16S Metagenomic Sequencing Library Preparation Manual (attached).
The Klindworth et al 2013 paper which Illumina based their primer sequences on is also attached."
# Data expected
'Data collection will take place from 01/03/2019 to 03/03/2019. The samples will be transported to CPGR on Monday, 04/03/2019. Results of the data analysis will be available within two weeks from then.'
# Bioinformatic analyses to be done
* Preprocessing of raw fastq files using the dada2 pipeline
* 'Alpha and beta diversity (Shannon diversity (H′) and Bray-Curtis dissimilarity index). Calculation of the relative abundance of each OTU per specimen. Fisher’s exact test for two-way tables. Construction of Log ratio biplots, dendograms and other illustrative (graphical) representation of data analysis.'
# Raw data
Raw (.fastq) copied from CPGR flashdisk to ilifu 26/6/2019
/ceph/cbio/users/katie/Levin/raw/
# Sample processing details from Pieter de Waal
'The Illumina® 16S metagenomics workflow will be used to analyze the three differently sourced milk types. Each sample will be analyzed in duplicate, producing a total number of six extracted libraries. A positive control will also be included in the analyses. The hypervariable V3 and V4 regions of the 16S ribosomal RNA gene (16S rRNA) will be amplified, using an Illumina® pre-designed primer pair. Barcoding for multiplexing of the samples will entail the use of the Nextera XT®-Index Kit.'
From Jeanne Korsman (Jeanne.Korsman@cpgr.org.za):
"16S Amplicon PCR Forward Primer = 5' TCGTCGGCAGCGTC _AGATGTGTATAAGAGACAG_ CCTACGGGNGGCWGCAG TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG
(Equivalent to the 16S "337F" primer)
16S Amplicon PCR Reverse Primer = 5' GTCTCGTGGGCTCGG _AGATGTGTATAAGAGACAG_ GACTACHVGGGTATCTAATCC GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC
(Equivalent to the 16S "800R" primer)
The underlined bases are the sequencing primer binding site, so these should not be present in any of the sequences. The sequences on the 5'end are where the barcodes are attached. They are also available in the 16S Metagenomic Sequencing Library Preparation Manual (attached).
The Klindworth et al 2013 paper which Illumina based their primer sequences on is also attached."
# Data expected
'Data collection will take place from 01/03/2019 to 03/03/2019. The samples will be transported to CPGR on Monday, 04/03/2019. Results of the data analysis will be available within two weeks from then.'
# Bioinformatic analyses to be done
* Preprocessing of raw fastq files using the dada2 pipeline
* 'Alpha and beta diversity (Shannon diversity (H′) and Bray-Curtis dissimilarity index). Calculation of the relative abundance of each OTU per specimen. Fisher’s exact test for two-way tables. Construction of Log ratio biplots, dendograms and other illustrative (graphical) representation of data analysis.'
# Raw data
Raw (.fastq) copied from CPGR flashdisk to ilifu 26/6/2019
/ceph/cbio/users/katie/Levin/raw/