Analysis
of primers couples for PCR amplification of SSU rRNA sequences
Rationale
: with the advent of 454 pyrosequencing technologies, it is now
feasible to sequences millions of sequences contained in a single
amplicon.
However, the tag length sequenced by the 454 is less than
400 nt even using the Titanium newer machine. As a result it is
necessary to develop or improve couples of primers that :
- Amplify
a short domain; indeed it is best to be able to retrieve the distal
primer, because differences in variable domains, length differences are
often as important as sequence variations.
- Amplify
a domain that has a good taxonomic resolution, both for discriminating
between species and to be sure that the same tags is not present in
very different taxa.
- Amplify a domain that the 454 can
sequence, because some secondary structures within rRNA sequences might
be difficult to sequence.
- Amplify specifically the required clade : i.e. Bacteria and
not Eukarya, or the reverse.
Analyses of 6972 couples of primers for amplications of Archaea, Bacteria and Protists SSU rRNA sequences : see results
Publication in preparation
We
have therefore compiled every SSU primer described in the litterature,
recovered 800,000 SSU sequences described in the public database and
longer than 500 nt.
We have also developed a very efficient program
(PrimerValidator) that can analyse extremely fast if a couple of primer
is able to amplify a given sequence. We did this test .ie. looking for every possible couple of primers in every known SSU sequence, allowing 1
difference between each primer and the target sequence, on top of the
degeneracies already present in a primer (if any).
On this web site you will find summary results for :
- How
many sequence each couple of primers can amplify in silico (at
one
difference possible), with mean amplicon size, mean positions of
amplicon in sequences (not exact numbers since sequences are of
different lengths).
- Sorted by decreasing abundances
- Sorted by primers sequences
- A file for Bacteria, Archaea, Protozoa (and
Viridiplantae), Metazoa, Organelles.
- For
some selected couples that seem to amplify well, a detailled taxonomic
description of their success or failure to amplify every higher taxon.
- ....
IMPORTANT
CONSIDERATIONS:
- The
numbers of public sequences (>=500nt) available for each clade
vary a lot
(Protozoa include Viridiplantae because they are very difficult to
sort):
- Bacteria : 671,593
- Archaea : 41,441
- Protozoa (including Fungi & Viridiplantae) :
75,908
- Organelles : 55,183
- Metazoa : NA yet
- If a primer is located near the 5" or 3" end, it may be
absent from many sequences, because:
- Lost of sequences are short
- If
the sequence was derived by PCR using these primers, they were in
theory removed from the submitted sequence (I will try to improve that
in the future)
List of primer containing papers.
This is an automated
PubMed qurey, download of full text papers, extraction of primers and
indexing when a primer described in a paper is also contained in an SSU
rRNA sequence (in construction).
Files that describe how many amplicons are obtained, when
screening the different divisions.
total
numbers of couples of primers read : numbers of different couples
analyzed (it is possible that the same couple was analyzed twice, as
for example testing a pair of F primer & F primer
inverted-complemented, and this F primer inverted-complemented was
found as a R primer in a different publication, but it is reported here only once).
number of different primers with amplicons : only this number produce
at least one amplicon
List provided by VC 1400
various primers
found in
PMID: 18047683 found in PMID:
18083874 See the synthesis of all 6972 pairs (in progress).
Preliminary analysis of published biodiversity studies
NOTE
: partial results using a list of "bacterial" primers provided by
Virginie Chapon. Metazoa
not analyzed yet
Bacteria
total numbers of couples of primers read 697
number of different primers with amplicons 351
Archaea
total numbers of couples of primers read 697
number of different primers with amplicons 281
Protists
total numbers of couples of primers read 697
number of different primers with amplicons 216
Metazoa
Organelles
total numbers of couples of primers read 697
number of different primers with amplicons 160
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Using
a list of 1400 couples, constructed from different sources.
Metazoa not analyzed yet
Bacteria
total numbers of couples of primers read 1400
number of different primers with amplicons 887
Archaea
total numbers of couples of primers read 1400
number of different primers with amplicons 804
Protists
total numbers of couples of primers read 1400
number of different primers with amplicons 559
Metazoa
Organelles
total numbers of couples of primers read 1400
number of different primers with amplicons 283
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described
in PMID: 18047683
Metazoa not analyzed yet
Bacteria
total numbers of couples of primers read 120
number of different primers with amplicons 73
Archaea
total numbers of couples of primers read 120
number of different primers with amplicons 48
Protists
total numbers of couples of primers read 120
number of different primers with amplicons 59
Metazoa
Organelles
total numbers of couples of primers read 120
number of different primers with amplicons 57
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described
in PMID: 18083874
Metazoa not analyzed yet
Bacteria
total numbers of couples of primers read 91
number of different primers with amplicons 60
Archaea
total numbers of couples of primers read 91
number of different primers with amplicons 67
Protists
total numbers of couples of primers read 91
number of different primers with amplicons 57
Metazoa
Organelles
total numbers of couples of primers read 91
number of different primers with amplicons 8
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Last updated by
Richard Christen 2009 May 18th
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If you are interested by a detailled study of a given primer pair,
please send me an email
Send email to christen use the mail server : unice.fr