Design: cDNA library was created by Vertis AG (Germany http://www.vertis-biotech.com/). Poly(A)+ RNA was purified from 10-20 µg of total RNA. Eight cercozoa libraries including a normalised one for Thaumatomonas oxoniensis were created as part of a larger set of 22 cDNA libraries constructed by Vertis AG. cDNA synthesis was according to Vertis' FLE (full-length enriched) protocol. First strand cDNA was synthesized using an oligo-d(T) adapter primer (5'-CCTTGGCTGTCACTCACTGCGA-d(T)25-3') and Maloney Murine Leukemia virusRNAse H reverse-transcriptase was used to extend the primer towards the 5'-end of the mRNA. After RNA hydrolysis, an adapter primer (5'-CTCTGGACCTTGGCTGTCACTCAGTT-3') was attached to the 3'-ends of the first strand cDNA, to allow directional cloning/sequencing of subsequent samples. The adapter sequences on both ends of the cDNA were then used to synthesize second strand cDNA and to amplify the cDNA with long and accurate (LA) PCR (Barnes 1994). To limit PCR induced bias only 18-21 PCR cycles were performed to amplify the cDNA. Between 3.3-5.3 µg of cDNA was thus created for each sulcozoan library, with had a size range of ~250-3000 nucleotides determined by gel electrophoresis. 454 pyrosequencing by the University of Liverpool’s Advanced Genomics Facility of these 22 libraries and two genomic sequence surveys used a multiplex sequence strategy on two separate half plates after tagging each of the 12 samples per run with a separate multiplex ID (MID) tag. For tagging, each cDNA from Vertis AG was blunt-ended and ligated by the Liverpool facility to one of the unique-decamers (MID tags). They computationally sorted the resulting sequences to assign each to the appropriate sample using Roche software. Fuller details of techniques are in the supplementary material of Cavalier-Smith, T., Chao, E.E., Snell, E.A., Berney, C., Fiore-Donno, A.M., Lewis, R., 2014. Multigene eukaryote phylogeny reveals the likely protozoan ancestors of opisthokonts (animals, fungi, choanozoans) and Amoebozoa. Mol. Phylogenet. Evol. in press DOI: 10.1016/j.ympev.2014.08.012. As explained there, some cross-contamination must have occurred between samples in a single run prior to or during tagging, so some sequences are not from the expected taxa. Therefore, users of these data are strongly advised to run single-gene phylogenetic trees to check the taxonomic origin of any sequence of interest, as we did for the 192 genes used for our published multigene trees, which were the primary purpose of the sequencing. To enable interpretation of possible cross-contamination the taxa and MID tags run on each plate were: Plate 1: MID1 - Thaumatomonas oxoniensis (Cercozoa) MID2 - Fabomonas tropica (Sulcozoa: Planomonadida) MID3 - Microheliella maris (Heliozoa) MID4 - Vannella simplex (Amoebozoa) MID5 - Ovalopodium desertum (Amoebozoa) MID6 - Nudifila producta (Cercozoa) MID7 - Thecamonas oxoniensis (Sulcozoa: Apusomonadida) MID8 - Mantamonas plastica (Sulcozoa: Mantamonadida) MID9 - Nutamonas howeae (Sulcozoa: Planomonadia) MID10 - Cercomonas clavideferens (Cercozoa) MID11 - Helkesimastix marina (Cercozoa) MID12 - Nolandella abertawensis (Amoebozoa) Plate 2: MID1 - Stenamoeba stenopodia (Amoebozoa) MID2 - Filamoeba sinensis (Amoebozoa) MID3 - Sandona ubiquita (Cercozoa) MID4 - Vexillifera bacillipedes (Amoebozoa) MID5 - Oxnerella micra (Heliozoa) + Minimassisteria diva (Cercozoa) MID6 - Flamella fluviatilis (Amoebozoa) MID7 - Cochliopodium minutoideum (Amoebozoa) MID8 - Multimonas media (Sulcozoa: Apusomonadida) MID9 - Ancyromonas sigmoides (=Planomonas mylnikovi) (Sulcozoa: Planomonadida) MID10 - Micrometopion nutans (Cercozoa) + Procryptobia sorokini (Euglenozoa) MID11 - Micronuclearia podoventralis (Sulcozoa: Varisulca: Rigifilida) genomic DNA MID12 - Rhogostoma minus (Cercozoa) genomic DNA Individual EST reads from each library were assembled using the 454 proprietary software, Newbler; contig construction parameters were chosen so primer sequences had no effect on contig assembly. Users should therefore remove primer sequences used in library construction from the raw submitted sequences.
Submitted by: University of Oxford
Sample:
cells of Thaumatomonas oxoniensisLibrary:
Name: Thaumatomonas oxoniensis
Instrument: 454 GS FLX Titanium
Strategy: EST
Source: TRANSCRIPTOMIC
Selection: PolyA
Layout: SINGLE
Spot descriptor:
5 forward
Pipeline:
show...hide...| Program | Version |
|---|
| Roche454 proprietary software for MID tag sequence removal and taxon assignment to each sequence |
| Newbler contig assembly | 2.0.00.20 |
Runs:
1 run, 40,643 spots, 23.6M bases, 51.1Mb