Sequencing

11th Annual Sequencing, Finishing, and Analysis in the Future Meeting
BUILDING REFERENCE MATERIALS FOR MIXED
MICROBIAL DETECTION WITH NEXT GENERATION
SEQUENCING
Friday, 3rd June 10:30 La Fonda Ballroom Talk (OS‐7.04)
Jason Kralj, Scott Jackson
National Institute of Standards and Technology
Myriad new sequencing and analytical technologies have emerged within the realm of metagenomic
sequencing. This has promised to transform the way clinical and environmental samples are analyzed.
Indeed, short (e.g. PGM Ion Torrent, MiSeq and HiSeq from Illumina) and long (Pacific Biosciences,
Nanopore) read platforms have much to offer in terms of ability to detect the faintest traces of pathogens
within a complex mixture of organisms. In addition, scores of post‐sequencing analysis software packages
offer a range of speed and sensitivity options to analyze the large data files, each with unique algorithms,
databases, and interfaces. All this choice presents a serious question—does this particular analysis set work
for my sample? While it is inconceivable that one could image the entire scope of experiments and sample
types for microbial systems, there are a few basic analyses that would lend confidence to the abilities of these
emerging tools.
We have endeavored to develop mixtures of microbial DNA with the purpose of identifying lim‐ its of
detection, contamination, bias, noise, and general strengths/weaknesses of various shotgun metagenomics
sequencing platfor Purified DNA represents an ideal condition that can be tightly controlled and analyzed,
without other confounding pre‐analytical variabilities such as extraction efficiency. Preliminary
experiments using NIST reference materials (RMs) and prospective RMs have allowed us to make idealized
mixtures of human, S. aureus, S. enterica subsp. enterica LT2,
P. aeruginosa, and C. sporogenes. Each well‐defined mixture (one equigenomic, the other a dilution
series) demonstrated the breadth of results from multiple analysis tools, and enable the end user to
recognize the limits of these syste
The results indicated that at DNA “concentrations” approaching 1:10 000 (in a background of
human DNA), or a few thousand total reads, detection was not challenging. However, accurate
identification highly depended upon the underlying database used for each tool. Furthermore,
some tools’ filtering algorithms accurately discriminate accurate reads from reads containing
significant numbers of sequencing errors, reducing the number of false positives. With this
knowledge, we’ve proposed in silico analyses that will utilize reference data sets to inform new
mixture designs that will provide benchmark analyses for the informatics tools. Meanwhile, we
are expanding our suite of organisms to provide greater breadth of genomic characteristics (e.g.
near neighbors, high/low G+C content, repetitive sequences) to stress test the sequencing
pipeline. Together, these will enable developers to better ascertain the capabilities of their
systems, and give regulatory agencies the tools and analyses needed to confidently evaluate the
new tools in mixed microbial detection.
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