Each sequence is identified by means of its sample tag and assigned to the file corresponding to the sample of origin by PyroClass. PyroMute then uses a number of quality filters to eliminate unreliable sequences. Sequence portions with a low Phred quality

score are removed.[21] Too-short sequences (<50 bp), including those generated by the previous filtering step, are also eliminated. Sequences are then aligned by means of a modified version of Smith-Waterman's algorithm,[22] in which the resolution of alignment matrices is accelerated and the identification of insertions and deletions (so-called indels) and the correction of length errors in homopolymeric sequences are improved. Quality filters subsequently remove Palbociclib purchase sequences with an identity score <80% relative to

the consensus sequence of the patient’s baseline sample. In the next step, an array of nucleotide substitutions and corresponding Phred quality scores is built and tested by means of a modified statistical test based on the binomial law[23] to eliminate sequences that are too rare and/or of poor quality, likely the result of sequencing errors. Finally, the remaining nucleotide sequences, considered reliable, BMN673 are converted into amino acid sequences and their respective frequencies are calculated. Each amino acid change is ascribed to its sequence of origin to subsequently analyze linkages between substitutions. PyroDyn uses data generated by PyroMute to detect and quantify increases or decreases in amino acid substitutions through mathematical modeling of their variations and correlation with an exponential growth model, which provided the best fit with the observed data. Briefly, in every patient, the frequency of each amino acid at each position is established at each time point. Assuming that HBV resistance is governed Meloxicam by exponential outgrowth of selected resistant variants, the best-fit curve is automatically drawn for each substitution

in each patient. Combined cut-off values have been established (r2 > 0.8 and growth rate > mean of the growth rates of all substitutions at all positions plus 2 standard deviations [SDs]) to differentiate significant exponential changes from polymorphism fluctuations. Finally, PyroLink has been designed to analyze genetic linkages between amino acid substitutions that have been selected by PyroDyn and to characterize the dynamics of viral populations bearing one or several amino acid substitutions over time. Briefly, linkages are automatically sought for every substitution identified with PyroDyn as exponentially growing or decreasing over time. Then, sequences that span all of the identified substitutions are extracted from PyroMute data and, when more than 100 such sequences are available, the proportion of sequences bearing no (WT), one, two, three, and so on, substitutions is calculated and used for subsequent analyses.