A straight-forward fake paired-end presentation into the assembler broke up the assembly due to short matches of product in various locations confusing the assembler.
Poly-A tails and any remaining vector sequences were then removed with TIGR's seqclean [ 51], traces identified as contaminants from E. coli or any of the pathogens used for stimulus were removed, and finally, all traces with less than 51 bases of quality sequence were discarded, resulting in 38,079 traces proceeding into the assembler.
In transcriptome assembly, the assembler frequently fails without the -cdna option because it expects approximately even coverage in genome assembly mode.
For the next two validation procedures, our intent was to more accurately represent the situation in which the assembler would be used.
Resulting sequences were assembled using the assembler in Geneious v5.1.7.
In the actual assembly step, the assembler used a strategy combining quality weighting (when there was not enough coverage depth) and simple majority (when deeper coverage was present) for contig consensus determination.
In most instances the assembler produces a set of contigs or scaffolds, which still leaves the genome in pieces.
The Trinity assembly was used as a reference sequence set to be appropriately refined and enriched, whenever possible, by a second de novo assembly performed with the assembler included in the CLC Genomic Workbench.
Comparisons of assembler performance have shown that assemblies will vary depending on the assembler used in terms of number of contigs generated, length of contigs, and resultant BLAST success [ 11, 19].
A trimming FASTA file, which includes polyA sequences of 5 bp, 10 bp and 20 bp in length, was used in conjunction with the vector-trimming option (-vt) of the assembler software, in order to trim off the polyA tails among the sequencing reads prior to assembly.
