To analyze the 16S rRNA gene sequence, quality filtering, including determination of the sequence length (>300 bp), end trimming, and determination of the number of ambiguous bases and the minimum quality score were performed using Trimmomatic (version 0.32).
The sequences were trimmed to remove primers and barcodes, quality filtered using sickle v1.33 with a minimum quality score of 20, assembled in mothur and aligned to SILVA 123 database.
As a measure of indel quality, we used the minimum quality score in the five flanking bases on either side of the indel and in any inserted bases.
Figures 3 and 4 display the relationship between the concordance and minimum quality score for the four scenarios in relation to the proportion of the population sequenced.
We experimented with various measures for identifying "low-quality" sequence (see Materials and Methods), but settled on the relatively simple measure of the minimum quality score observed within a 5 base pair distance of the indel.
For imputation of CA, the results were similar between the three methods for imputation, with Scenarios 1 and 2 producing the best concordance and Scenarios 1 and 3 producing the highest mean and minimum quality score.
Throughout the pipeline described herein, we have had to make decisions about acceptable minimum quality scores for calling variants, as well as many other important assembly and mapping parameters.
For AhLOX7, seven out of the top ten predicted mutations had minimum quality scores above 19, and the minimum variance multipliers around 12. We selected six sets (minimum quality 19, 20, 21, with minimum variance multipliers 12 and 14, and the minimum read percentage with non-reference nucleotide of 0.05%) as candidate mutants.
For each execution mode the user can specify the minimum base quality score, the minimum read quality score and the minimum depth of coverage for the pileup computations and the significance threshold for the statistical tests used in the GENOTYPE and ASE modes (default values set to 0, 0, 1, 5%, and 5%, respectively).
Assembly parameters were as follows: file format, BAM; mer Size, 21; mer skip query, 2; minimum match percentage, 93; maximum gap size, 6; minimum aligned length, 35; match score, 10; mismatch penalty, 20; gap penalty, 30; SNP calculation method, diploid bayesian; minimum SNP percentage, 5; SNP confidence threshold, 10; minimum SNP count, 2; minimum base quality score, 5.
Parameters for SNP detection included: a minimum read coverage at the potential SNP of 6, window length of 9, minimum average quality score of 15 and minimum central quality (i.e. quality of the SNP base) of 20.
