In addition, the quality filtered raw sequence reads were mapped using bwa-0.5.9 [ 90] and BLASTN against the CDS sequences of lin gene database with a minimum coverage cut-off = 100X [ 16].
In total, 18,894 RNA-derived contigs were assembled (comprising 22.1 Mbp of transcriptome sequence) that were greater than 200 bp in length (mean = 1170 bp, Figure 1 and Additional file 2), with a median coverage per base (CPB) per contig of 37×, ranging from 8× (minimum coverage cut-off for assembly) to 5,262× (Additional file 1 Figure S2).
Additional filters should therefore be applied when validating the mismatches at a particular genome position, such as minimum coverage cut-offs, and validation against the number of mismatches expected given the sample processing procedure.
The appropriate k-mer coverage cut off was determined using an R package plotrix (Additional file 4).
First, the sequences were filtered using a minimum quality cut off value of 40.
Expect values (E values) of 0.001 and bit scores of 30 were used as the minimum allowed cut off.
These two data sets were used to mine the protein sets of each sequenced Lachnospiraceae genome using USEARCH 4.0.38 (Edgar 2010) with an e-value cut off of 10− and a minimum identity cut off of 70%.
A BLAST search of all contigs in the high coverage dataset (7,325 sequences) produced 3,788 positive hits (BLASTx search of the NCBI nr database, minimum e-value cut off = 10-6; average e-value = 3.4 × 10-9; Additional file 2).
As a minimum cut off for group comparisons, and 0.9 for individual comparisons.
Approximately 40 million Solexa (Illumina Inc., San Diego, CA, USA) sequenced ESTs were also included from the 'digital transcriptome' of B. napus lines TapidorDH and Ningyou7 [24], which was assembled using Velvet [25] with minimum contig length of 100 bp, coverage cut-off of five and k-mer value of 23, producing 29,956 contigs.
The results of the Velvet assemblies were then run through Oases using an insert length of 300 bp, a minimum transcript length of 200 bp, a coverage cut-off of 5, and an edge fraction cut-off of 0.25.
