Pacific Biosciences data analysis-(Published October 15, 2013).
A microbial clock provides an accurate estimate of the postmortem interval in a mouse model system
We took advantages of the long reads provided by the Pacific Biosciences (PacBio) sequencing platform to gain more detailed taxonomic resolution of the abundant bacteria and eukaryotes found in the early and late stage decomposition communities. In both cases high quality circular consensus sequences (CCS) were used. The 16S PacBio data consists of sequences of 800–900 bp in length. The taxonomic identity of the sequences was assessed by assigning sequences to OTUs using the Greengenes February 2011 release of the Greengenes (DeSantis et al., 2006; McDonald et al., 2012) 97% reference dataset. Taxonomy assignments of the long PacBio reads agreed with the short Illumina reads at the family level and the long reads made is possible to determine the taxonomic identity of the reads to the genus and in most cases species level. Taxonomy of the PacBio sequences was also verified by placing sequences within a phylogenetic tree using RAxML EPA algorithm (Berger et al., 2011). The 18S PacBio sequences were roughly 1200 base pairs in length. The 18S data was clustered into OTUs at 97% similarity using the open-reference protocol described above and the curated Silva 108 database. Initial taxonomy assignment was done by BLAST (Altschul et al., 1990) with an e-value threshold of e-10. Taxonomy assignment was refined by placing the sequences within the Silva 108 reference tree using maximum likelihood with RAxML EPA. These resulting taxonomy assignments were used to resolve the taxonomy of highly abundant community members. Bacterial and microbial eukaryotic taxa found were sorted by abundance at each site are reported in Supplementary File 1B. Genus and species level taxonomy was reported in relevant text of the manuscript. Because PacBio data were generated from only a small subset of samples we did not use these data for comparative analyses, Go to IHUB M B,for link)