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Challenges posed by aligning reads, as well as the impact of reference biases on diversity estimates are also discussed.
Next-generation sequencing projects commonly commence by aligning reads to a reference genome assembly.
Positions with only one aligned read were ignored.
The aligned read files were processed by Cufflinks v2.0.2 [ 28].
To avoid possible PCR artifacts, no more than one uniquely aligned read per strand was kept.
Then the first and last five bases of each aligned read were removed.
Nucleotide sites with coverage less than two were filtered from the aligned read data set.
We manually inspected the aligned reads onto miRNA loci and removed non-specific alignments.
Quality control metrics should be calculated for raw reads, as well as for aligned reads.
Testes show 166 reads per million aligned reads.
Aligned read frequencies (per million aligned reads) were calculated for each sample and locus using Alpheus® [48].
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