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assessment of a repetitive sequence across more than 2200 microbes, the researchers pre- dicted that SMRT Sequencing could be used to automatically close at least 70% of known bacteria and archaea genomes. This could be crucial during a pandemic or foodborne out- break, where correctly and rapidly identifying a microbial strain can inform the choice of treatment as well as epidemiological studies.
Long reads plus automated sizing While long-read sequencing on its own can
produce impressive assemblies, scientists have found that the use of automated DNA size selection to remove smaller fragments prior to sequencing has a marked impact on generated read length. By pairing BluePippin size selection from Sage Science (Beverly, Mass.) with SMRT Sequencing, the average length of sequenced DNA fragments has been doubled in some cases, creating as- semblies with unprecedented contiguity and accuracy. In an effort to improve the quality of the human reference genome, scientists at the University of Washington (Seattle, Wash.) used BluePippin-sized PacBio sequence data to close or shrink more than half of the remaining gaps in the assembly, most of which included highly repetitive sequence.4
Scientists at the Icahn School of Medicine at Mount Sinai, Cold Spring Harbor Laboratory (Cold Spring Harbor, N.Y.) and European Molecular Biology Laboratory (Heidelberg, Germany) conducted the first analysis of a diploid human genome using single molecule technologies, including SMRT Sequencing, and produced “the most contiguous clone-free human genome assembly to date.”5
The study
focused on the well-characterized NA12878 genome and produced an assembly with scaf- fold N50 values close to 30 Mb. Scientists used the BluePippin platform to size-select libraries, removing fragments smaller than 7 kb, prior to sequencing with the PacBio system. “Without selection, smaller 2000–7000 bp molecules dominate the zero-mode waveguide loading distribution, decreasing the sub-read length” that can be achieved with the sequencer, the authors noted.
In separate work, researchers from the Norwegian Sequencing Centre (Oslo, Norway) ran several libraries on the PacBio platform
with and without BluePippin sizing to deter- mine the impact of size selection.6
In one test,
the average DNA insert length doubled from 3000 bases to 6000 (in the years since this analysis was done, typical DNA insert lengths sequenced have increased to about 10 kb with- out size selection and 15 kb with size selection). Without size selection, 50% of bases sequenced in this project were in reads 5 kb or smaller; after size selection, half of all bases were in reads of at least 10 kb.
Looking ahead As advances continue for long-read sequencing and automated DNA size selection, scientists can expect read lengths to increase even more. That will allow for the automated completion and closure of even larger genomes, leading to pushbutton reference genomes. Ultimately, it will be feasible to generate platinum-quality genomes that will enable researchers to inves- tigate the full universe of genetic diversity in any species.
References
1. Loomis, E.; Eid, J. et al. http://genome.cshlp. org/content/23/1/121.full
2. Ritz, A.; Bashir, A. et al. http://bioinformatics.
oxfordjournals.org/content/30/24/3458.full
3. Koren, S.; Harhay, G.P. et al. http://www.
genomebiology.com/2013/ 14/9/R101
4. Chaisson, M.J.P.; Huddleston, J. et al. http://
www.ncbi.nlm.nih.gov/pmc/articles/ PMC4317254/
5. Pendleton, M.; Robert Sebra, et al. http://
www.nature.com/nmeth/journal/v12/n8/ full/
nmeth.3454.html
6. Nederbragt, L. https://flxlexblog.wordpress. com/2013/06/19/longing-for-the-longest- reads-pacbio-and-bluepippin/
T. Chris Boles, Ph.D., is chief scientific officer, Sage Science, Inc., Beverly, Mass., U.S.A. Jonas Korlach, Ph.D., is chief scientific officer, Pacific Biosciences, 1380 Willow Rd., Menlo Park, Calif. 94025, U.S.A; tel.: 650-521-8000; e-mail: jkorlach@
pacificbiosciences.com.
AMERICAN LABORATORY 43 MARCH 2016
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