16S_PCR_Sequence_Analysis

Open Serial Cloner.

Software

Value	Label
Serial Cloner	NAME
Franck Perez	DEVELOPER
http://serialbasics.free.fr/Serial_Cloner.html	LINK
2.6.1	VERSION

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.

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

Value	Label
FinchTV	NAME
macOS	OS_NAME
Geospiza Research Team	DEVELOPER
https://digitalworldbiology.com/FinchTV	LINK
1.5.0	VERSION

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).