For eight of the 16 repeated subtype C samples, the proportion of reads with K65R was >1.0% in each of the duplicate runs and for seven of the 16 repeated subtype C samples, the proportion of reads with K65R was <1.0% on both runs.
K65R was detected in at least 1.0% of reads in 8/18 (44%) subtype C samples and in 1/27 (3.7%) subtype B samples (p = 0.01; Fisher's Exact Test).
Among the subtype C samples, K65R was detected in at least 1.0% of reads in eight samples, including 7 of 11 with the most common subtype C pattern and the one sample with the second-most common subtype C pattern.
Among the subtype B samples, K65R was detected in more than 1.0% of reads in a sample with a complex mixture of KKK nucleotide variants including AAA-AAG-AAA (the second-most common subtype C pattern) in 38% of reads, AAG-AAA-AAA (the most common subtype B pattern) in 7% of reads, and an uncommon pattern AAG-AAG-AAA in 50% of reads.
For plasmid clones with a subtype C nucleotide pattern, UDPS detected K65R in a mean 1.1% of reads (range 0.7 1.3).
In contrast, the four clones with the two most common subtype B KKK patterns had K65R in a median of 0.15% of reads (range 0.1% 0.3%; P = 0.007; Wilcoxan Rank Sum Test).
The subtype from which the surrounding viral sequence was derived did not significantly influence the number of reads with K65R.
Of the four subtype C samples with the most commonly reported subtype B KKK nucleotide variant (AAG-AAA-AAA), none had K65R in 1.0% or more of reads.
In nasopharyngeal samples, although whole genome analysis was not available because the majority (>90%) of reads were host genome derived, 20 460 Flu-reads were detected, which was sufficient for subtype identification.
Using the relative proportion of the total unique read counts against total number of reads, a condition tree (Genespring 7.3.1, Agilent) of all novel candidates plus known miRNAs gave good separation of the different histological subtypes of sample (Figure 3).
