HBV amplicon sequencing - PacificBiosciences/DevNet GitHub Wiki

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)