Regarding the choice of Ref database & a related Question

Commands in mothur
1

I created my custom silva.v4.fasta from silva.full_v123.fasta instead of silva.bacteria.fasta mentioned in MiSeq SOP.

I made silva.full_v123.fasta by following instructions provided in:
http://blog.mothur.org/2015/12/03/SILVA-v123-reference-files/

This is because I wanted to include both Archaea as well as Bacteria.

I just wanted to make sure I am on the right track because we have to analyse some environmental samples – that can include diverse organisms. Is my approach correct?

=== Question 2

By the way, similar to MiSeq SOP (an excellent resource) for mapping taxonomy from 16S data for bacteria and archaea, do we have a similar resource for 18S taxonomy analysis.

This is because in our project we wanted to identify all components of an environmental sample like:
bacteria, fungi, algae, protozoa,…

Please suggest. Thank you in advance.

2

one of the silva db should contain all domains (can’t remember off the top of my head which one). What primers are you using that hit all 3 domains? Hugenholtz worked on developing some when he was at JGI but they missed big groups of Archaea and were for v9.

3

Adding to my question above, for example, what could be a comprehensive reference database for eukaryotes (or how to construct one), which can be used in mothur analysis. Thank you.

4

Primers I use are the following:

All sequences 5’-3’ orientation:

16S V4 Forward :: GTGCCAGCMGCCGCGGTAA
16S V4 Reverse :: GGACTACHVGGGTWTCTAAT

And for the 18S ITS primers:
18SITSF :: TCCGTAGGTGAACCTGCGG
18SITSR :: TCCTCCGCTTATTGATATGC

Thanks for your help.