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HBV amplicon sequencing
Instrument: PacBio RS
Chemistry: C2
Enzyme: C2
We examined detection sensitivity of SMRT sequencing to detect very low level mixtures of known resistance-associated mutations in the Polymerase Gene (P) of HBV. Three mutations were introduced into HBV plasmids by site-directed mutagenesis. Two amplicons (575 bp and 1,389bp) were generated for both wildtype and mutant plasmids, which were purified, normalized, and mixed by serial dilution at 20%, 10%, 5%, 2.5%, 1.25%, 0.62%, 0.31%, 0.16%, and 0.08% frequencies. SMRTbell™ libraries were constructed and sequenced using standard Pacific Biosciences® C2 chemistry and protocols.
Abbreviations have been used in all the file names to identify the sample mix and the following table shows the mix code:
======================= % Mut 1389bp 575bp ======================= 0% 1L 1S 0.08% 2L 2S 0.16% 3L 3S 0.31% 4L 4S 0.63% 5L 5S 1.25% 6L 6S 2.50% 7L 7S 5% 8L 8S 10% 9L 9S 20% 10L 10S 100% 11L 11S ========================= For example: * m45min_575bp_11s_aligned_reads.sam ===> 2 * 45 min movie, 575bp amplicon, 11S (100% mutant mix sample) * m90min_1389bp_9l_aligned_reads.sam ===> 1 * 90 min movie, 1389bp amplicon, 9L (10% mutant mix sample) ### Download Datasets Final (Secondary Analysis) Data * [HBV 575bp amplicon](http://files.pacb.com/datasets/secondary-analysis/hbv_amplicons/hbv_575bp_alignment_files.tar.gz) (125 MB) includes one alignment file in SAM format for each mix; 2 * 45 min movies * [HBV 1389bp amplicon](http://files.pacb.com/datasets/secondary-analysis/hbv_amplicons/hbv_1389bp_alignment_files.tar.gz) (93 MB) includes one alignment file in SAM format for each mix; 1 * 90 min movies Reference sequence * [HBV 575bp amplicon](http://files.pacb.com/datasets/secondary-analysis/hbv_amplicons/cat_wild_short.fasta) * [HBV 1389bp amplicon](http://files.pacb.com/datasets/secondary-analysis/hbv_amplicons/cat_wild_large.fasta)
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