16S rRNA gene marker 16S rRNA gene marker intra-gene variability intra-gene variability primer selection primer selection size & information size & information content content Primer selection, information content, alignment and length Primer selection, information content, alignment and length
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16S rRNA gene marker intra-gene variability primer selection size & information content Primer selection, information content, alignment and length.
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16S rRNA gene marker 16S rRNA gene marker
intra-gene variabilityintra-gene variability
primer selectionprimer selection
size & information contentsize & information content
Primer selection, information content, alignment and lengthPrimer selection, information content, alignment and length
Universality depends on the known datasetUniversality depends on the known dataset
Different phyla may have differences in the Different phyla may have differences in the ““universaluniversal”” regions (e.g. EUB 338) regions (e.g. EUB 338)
Primers used for rRNA cloning may give biased resultsPrimers used for rRNA cloning may give biased results
Metagenomics without amplification steps may reveal hidden diversityMetagenomics without amplification steps may reveal hidden diversity
EUB338 IEUB338 I Most Most BacteriaBacteria GCTGCCTCCCGTAGGAGTGCTGCCTCCCGTAGGAGT
EUB338 IIEUB338 II PlanctomycetalesPlanctomycetales GCGCAAGCCGCCAACCCGTAGGCCCGTAGGTTGTGT
EUB338 IIIEUB338 III VerrucomicrobialesVerrucomicrobiales GCTGCCGCTGCCAACCCGTAGGCCCGTAGGTTGTGT
Daims et al. 1999. System Appl Microbiol 22, 434-444
Primer selection Primer selection size of the amplicon size of the amplicon
GM38
616Valt 8
GM5 GM5-clamp
341
518F518
518R518
GM41492
907R907
945F945
Bac1055F1055
630R1529
S1505
ideally the almost complete gene (ideally the almost complete gene (~ 1520 nucleotides) should be sequenced~ 1520 nucleotides) should be sequenced
many amplifications skip sequencing the helix 50 (~ 1490 nucleotides)many amplifications skip sequencing the helix 50 (~ 1490 nucleotides)
many clone libraries are based on just partial amplicons (~ 900 nucleotides)many clone libraries are based on just partial amplicons (~ 900 nucleotides)
Pairs GM3 (8) – GM4 (1492) most widely usedPairs GM3 (8) – GM4 (1492) most widely used
16S rRNA sequencing has grown exponentially in parallel to the development of sequencing techniques
Yarza et al., Nature Revs. 2014. 12: 635-645 Tamames & Rosselló-Móra 2012 TIM 20:514-516
rRNA cataloguing
radioactive Sanger
sequencing
non-radioactive
Sanger sequencing
reverse transcription sequencing
NSG
The database is exponentially increasing99% environmental sequences
1% cultured organisms
3.8 x106 sequences700,000 / year (last three)
Sources of sequences and quality
rRNA Cataloguing (up to late 80’s), bad quality
reverse transcription sequencing (up to late 90’s), bad quality
Sanger methods (radioactive, biotin-labelled, terminal-dye… still in use)
cloning DNA, good quality
direct amplification, good quality
DGGE/TGGE, short sequences, bad quality
NSG, short sequences
454 technology (now up to 800nuc, mean of 500nuc), moderate quality
illumina (now 2x 250nuc), too short
16S rRNA sequencing has grown exponentially in parallel to the development of sequencing techniques
Quast et al., 2013, Nuc Acid Res. 41: D590-D596
www.arb-silva.de
SILVA release 119 (July 2014)
rate of rejection of about 30% of the existing sequences
short sequences are generally worse than long stretches