Detail of the upgraded genome
Genomic features of C. sinensis
Besides paired-end data previously published [1], we sequenced two
paired-end and two mate-pair libraries again. In total, 263.38 million raw
pairs of sequence reads (107X coverage) were produced，with 221.62
million (63X coverage) used for genome assembly after data filtering
(Table S1). The assembled genome includes 4,348 scaffolds with a total
length of 550 Mb. The upgraded version, with an N50 scaffold/contig
length of more than 417/233 kb and the largest scaffold of more than 2
Mb, was improved compared with the previously published version
(Table 1). The GC content was calculated to be approximately 43%
(Figure S4). In C. sinensis, approximately 32% of the genome represents
interspersed repeats, based on both known and ab initio repeat libraries
(Figure S5). To estimate the gene coverage of the assembly, CEGMA core
genes [2] and assembled transcript data were mapped to the C. sinensis
genome. As a result, approximately 97% of the transcripts were mapped,
and on average, approximately 83% of the assembled transcripts were
mapped to the same scaffold with at least 90% of their total length (Table
S14).
Three methods, including similarity-based, ab initio methods and
genome-guided assembly of RNA-Seq, were applied to identify
protein-coding genes. After manual verification, a total of 13,634 gene
models were retained as the final gene set. Detailed analysis of gene
length, exon number per gene and gene density in C. sinensis showed
similar patterns to those seen in both S. japonicum and S. mansoni (Table
2). Approximately 76.6% of the genes have homologs in the NCBI
non-redundant database, and 58.8% can be classified using Gene
Ontology terms. Overall, 79.6% of the genes could be annotated (Table
S2).
In addition, non-coding RNAs were identified by the methods
mentioned in our previous study [1]. Nine rRNA fragments, 276 tRNAs,
482 small nucleolar RNAs, 149 small nuclear RNAs and 165 miRNA
precursor genes were identified in the C. sinensis genome (Table S14),
and 107 miRNAs had been found expressed previously [3] (Table S15).
Methods of assembly and annotation
DNA library construction and sequence analysis
Besides paired-end data previously published [1], we sequenced two
paired-end and two mate-pair libraries again. The two short-insert (400and 550-bp) DNA libraries were constructed from the same fluke used in
our previous study [1] as described in the Paired-End Sample Preparation
Guide (Illumina, San Diego, CA, USA). In addition, the two long-insert
(2000- and 5000-bp) DNA libraries were constructed from twenty adult
worms that were pooled together using the SOLiD Mate-Paired Library
Construction Kit.
Cluster generation was performed on the cBot (Illumina), following the
cBot User Guide. A paired-end sequencing run was then performed on the
Genome Analyzer IIx (Illumina) as described in the Genome Analyzer IIx
User Guide (Illumina). After masking adaptor sequences, removing
contaminated reads and trimming low-quality reads, the cleaned data
were processed for computational analysis.
Genome assembly and repeat identification
The whole genome shotgun sequencing raw data were filtered using
Fastx-tools [4][13] using the following criteria: 1) reads containing
sequencing adaptors were removed; 2) reads were trimmed to 103bp for
Celera Assembler limitation; 3) reads with low-quality (the percentage of
nucletides with Q20 score were lower than 50%) were removed; and 4)
artificial reads were removed.
The Celera Assembler v6.1 [5] was used to assemble contigs and
construct scaffolds with the mate-pair data using SSPACE [6]. Finally, the
gaps were filled with GapCloser [7][16].
Known repetitive elements were identified using RepeatMasker [8,9]
with the Repbase database [10,11] (version: 2009-06). A de novo repeat
library was constructed using RepeatModeler [12,13], and default
parameters were then used to generate consensus sequences and
classification information for each repeat family. RepeatMasker was
again run on the genome using the repeat library built with
RepeatModeler.
Gene prediction
Predicted proteins from S. japonicum and S. mansoni were aligned to the
C. Sinensis transcriptome to identify conserved genes. Because the
GeneWise [14] program is time consuming, proteins from schistosome
were first aligned with the C. sinensis genome using genBlastA [15].
Subsequently, matched genomic regions were extracted and GeneWise
was used to identify exon/intron boundaries. The Program to Assemble
Spliced Alignments (PASA) [16] was used to generate spliced alignments
of putative full-length cDNAs to the unmasked assembly, which was then
used to train the ab initio gene prediction software, Augustus [17].
Genscan [18] was run using the model parameters for human. The
RNA-Seq data from the four tissues were aligned to the genome using
TopHat [19]. Cufflinks [20] was then used to assemble the transcripts
with junction information. Gene predictions were generated using
Augustus and Genscan, and spliced alignments of S. japonicum and S.
mansoni proteins and transcripts produced by Cufflinks were integrated
with EvidenceModeler [21]. Transcript isoforms were constructed by
Cufflinks with -g and default parameters, followed by Cuffcompare.
Alternative splicing events were predicted using altSpliceFinder program
in Ensembl API tools.
Protein domain analysis
InterProScan [22] was run on all species (C. sinensis, S. japonicum, S.
mansoni, C. elegans, D. melanogaster, D. rerio, G. gallus and H. sapiens)
to predict protein sequences. Matched sequences tagged as ‘True Positive’
(status ‘T’) by InterProScan were retained.
Functional annotation
C. sinensis reference genes were mapped to KEGG pathways [23] by
BLAST (e-value<1e-5). BLAST searches against the Swiss-Prot database
and NCBI non-redundant database (e-value<1e-5) were conducted to
provide comprehensive functional annotation.
CEGMA validation
Using default parameters, the CEGMA [2] set of 458 core eukaryotic
genes was used to evaluate the completeness of the predicted gene
models using the GenBlastA [15] program.
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