Dec 12, 2022
16S_PCR_Sequence_Analysis
- 1Rutgers University
Protocol Citation: Kenneth Acosta 2022. 16S_PCR_Sequence_Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr49e3gmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 09, 2022
Last Modified: December 20, 2022
Protocol Integer ID: 73758
Keywords: Genotyping
Abstract
Protocol to generate a consensus 16S rRNA gene sequence from forward and reverse sequences.
Materials
Software
Serial Cloner
NAME
Franck Perez
DEVELOPER
Software
FinchTV
NAME
macOS
OS
Geospiza Research Team
DEVELOPER
Adding U515 sequence feature
Adding U515 sequence feature
Open Serial Cloner.
Software
Serial Cloner
NAME
Franck Perez
DEVELOPER
Go to “Features” → “Manage Features”.
Add new collection. Name “16S rRNA Gene Conserved Regions”.
Add new feature. Name “U515”. Add sequence “GTGCCAGCAGCCGCGGTAA”.
Check “Scan For This Feature” box. Click Ok.
Generating consensus 16S rNA gene sequence
Generating consensus 16S rNA gene sequence
Upload 16S rRNA gene forward and reverse sequences for the sample.
Go to “Function” → “Align Two Sequences”.
Check “anti-parallel” box for reverse sequence.
Click “Local Align”.
Click “Build Consensus”. New dialog box will appear.
Select “Unsaved Consensus” window then click “Features” → “Scan Sequence”. “U515” feature should be highlighted in consensus sequence.
Click “Features” tab in the “Unsaved Consensus” window.
The nucleotide positions of the “U515” marker will be given. Select the “from” box in top right corner of window and type in the nucleotide position that is -385 nt from the beginning of the “U515” marker. Select the “to” box in top right corner of window and type in the nucleotide position that is +216 nt from the end of the “U515” marker. A 620 bp consensus sequence should be generated.
To check for quality of consensus sequence select “Align Two Sequences” window and look for differences in base calls between forward and reverse sequences within the consensus sequence. Sometimes there will be a mismatch or gap between forward and reverse sequences. To assist in making the right base call at a certain nucleotide position use FinchTV to view chromatograms and determine the right call using the higher-quality base call.
Software
FinchTV
NAME
macOS
OS
Geospiza Research Team
DEVELOPER
Save consensus 16S rRNA gene sequence.
Annotate consensus 16S rRNA gene sequence by searching a database (e.g. NCBI) or using a 16S rRNA gene classifier (e.g. RDP classifier).