Genome analyzer system

The cDNA library was prepared using the TruSeq^TM RNA Sample Preparation Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Poly(A)-containing mRNA was purified by Oligo(dT) magnetic beads from 10 μg total RNA sample and fragmented using divalent cations. The cleaved RNA fragments were used for the first strand cDNA synthesis using reverse transcriptase and random primers, followed by second strand cDNA synthesis using DNA polymerase I and RNase H. After second strain cDNA synthesis, fragments were treated with end repair, A-base tailing and adapter ligation consecutively. The sample was further treated by gel size fractionation and PCR amplification to create final cDNA library. The cDNA library was sequenced on the Illumina Cluster Station and Illumina Genome Analyzer system according to the manufacturer’s instructions. The Trinity method was used for de novo assembly of Illumina reads of T. tridentatus embryos [27 (link)]. Briefly, the trinity using de Bruijn graph algorithm was run on the paired-end sequences with the fixed default k-mer size of 25, minimum contig length of 200 and paired fragment length of 500.

The Illumina Genome Analyzer System generated 61,593,058 paired-end reads of 100 nt with an insert size of 455 nt. The 454 run generated 78,087 single-end reads with an average length of 190 nt.
Velvet (Zerbino and Birney 2008 (link)) was used to make a hybrid assembly with the Illumina and 454 reads generating 118 contigs with an N50 of 231,364. All contigs were compared with the nt database of NCBI using the BlastN algorithm and only two contigs had high-scoring alignments with sequences from Flavobacteria.
A second assembly was run using Phrap (Gordon et al. 1998 (link)) taking the velvet contigs as input, generating a single circular contig of 309,299 bp which corresponded to the chromosome sequence of the flavobacterial endosymbiont. Average coverage per nucleotide was 1571.4×. Protein-coding genes were predicted using Glimmer3, GeneMark.hmm, and Blast; tRNAs and a tmRNA were identified with tRNAscan-SE; and rRNAs were identified using the web version of WU-BLAST against the Rfam 11.0 sequence library.
Gene function annotation of the predicted protein-coding genes was based on results of BlastP searches against the RefSeq database and of hidden Markov model searches of the Pfam and TIGRFAM databases. The GenePRIMP pipeline (Pati et al. 2010 (link)) was used to search for gene call anomalies and the resulting report was used to perform manual curation of the genome.

Total RNA samples were extracted from the wild type CAI4, the gdt1/gdt1, the pmr1/pmr1, and the gdt1/gdt1 pmr1/pmr1 mutants grown to log phase in SD-URA at 30 °C. RNA integrity was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). RNA-seq libraries were constructed using Illumina’s TruSeq RNA Sample Preparation Kit (Illumina Inc., USA). RNA sequencing, data analysis and sequence assembly were performed by Beijing BioMarker Technologies (Beijing, China). Preparation of the paired-end libraries and sequencing were performed following standard Illumina methods and protocols. The mRNA-seq library was sequenced on the Illumina Hiseq 2500 and Illumina Genome Analyzer system.
To obtain high-quality clean read data for de novo assembly, raw reads from mRNA sequencing were filtered by discarding the reads with adaptor contamination, masking low-quality reads with ambiguous ‘N’ bases and removing reads in which more than 10% bases had a Q-value < 20. Clean reads were assembled into full-length transcriptome from RNA-Seq data with the reference genome (http://www.candidagenome.org/).