Interview: DNA Sequencing

Genome Center Offers NGS Applications toSupport Microbiome Studies
Dr. Thomas Sutter selected the Illumina MiSeq® System and Nextera® XT kits to expand thecapabilities of the University of Memphis genome center.
Thomas Sutter, Ph.D., is the Director of the W. Harry Feinestone
Center for Genomic Research and the Feinestone Chair of Molecular
Biology at the University of Memphis. He conducts research in thearea of cancer causation and prevention in addition to running theuniversity’s genome center. Dr. Sutter opened the center with the goalof making a broad base of technology accessible to investigators
across the campus. Last year, Dr. Sutter wanted to develop nextgeneration sequencing (NGS)-based genomic applications and data
analysis that would assist researchers with discovery and in obtaining
grant funding.
A typical project for the genome center involves resequencing small
genomes (~5 Mb) such as microbiome samples and PCR amplicons.
Some bacterial strains are difficult to culture making it challenging
to obtain sufficient DNA for sequencing. Others are notorious forhigh nucleotide repeat regions, which can introduce bias in library
preparation kits requiring enzymatic fragmentation. “We wanted asystem that ensured correct loading of high-quality DNA and one thatdelivered the highest-quality results,” explains Dr. Sutter.
Dr. Sutter and his colleagues evaluated the Illumina MiSeq® Systemand the Nextera® XT DNA Library Preparation kits. The MiSeq
System’s industry-leading data quality, accuracy, and streamlined
workflows are ideal for resequencing small genomes and amplicons.
When combined with Nextera XT kit’s rapid library preparation andminimum hands-on time, sequencing results can be obtained in justa day and a half. By implementing these Illumina technologies,
Dr. Sutter found that he progressed faster in reaching his goals thanhe expected.
Q: What challenges did you face when introducing NGS to thegenome center?
Thomas Sutter (TS): Before we moved to NGS, we were mainly amicroarray facility conducting gene expression analysis. It soon became
clear that the field of genomics was moving to NGS. For our center
to thrive, it was imperative that we quickly adapt NGS technology tomeet the diverse needs of our researchers and center members, whorange from evolutionary biologists to biomedical engineers. We wanted
to provide applications to address a multitude of needs, and assist ourinvestigators in obtaining grant funding.

Thomas Sutter, Ph.D., is the Director of the W. Harry Feinestone Center
for Genomic Research and the Feinestone Chair of Molecular Biology
at the University of Memphis in Tennessee. With the MiSeq System andthe Nextera XT DNA Library Preparation kit, NGS technologies wereintroduced into the university’s genome center in less than a week.

to develop all of the applications that the MiSeq can offer, we also liked
that Illumina products provide a convenient, one-stop shop for NGS
applications. The Illumina platform continues to deliver expanding readlengths and mate pair capabilities along with its ease-of-use. I believethat we made the right choice.
Q: How long did it take you to implement the Illumina
NGS platform?
TS: The transition to NGS went very smoothly. The Nextera XT kit
was introduced when we bought the MiSeq System. The Illumina
field application scientist (FAS) came in for a two-day training tohelp get us started. The following week, we were sequencing
2 million base pair bacterial genomes. In just two days, we were up
and running. The MiSeq has given us a good opportunity to learn
the Illumina chemistry, become proficient at library construction,and build the back end for data analysis and storage.

Q: Why did you decide to go with the MiSeq System?
TS: We were new to the NGS area and wanted a system that we could
learn to use and implement quickly. We looked at all of the existing
technologies and wanted to avoid having to combine different platforms
to get the output we desired. We chose the MiSeq System mainly forthe quality of its reads and the high Q30 values. Because we wanted“Nextera XT allows our investigators
to spend less time making libraries
and more time making discoveries.”

Interview: DNA Sequencing
Q: What features led you to choose Nextera XT
library preparation?
TS: There are several features that attracted us to Nextera XT. The
accommodation of low DNA input down to 1 ng was of particularinterest to us. No matter how easy it is to obtain samples, they’realways precious and we never want to use more than we have to.
We particularly liked the Nextera bead-based normalization. The
bead step eliminates the real-time PCR step and the validation witha Bioanalyzer that’s necessary for normalizing the samples to ensure
proper DNA loading and high-quality runs. With Nextera XT, when
we load bar-coded samples onto the MiSeq flow cell, the reads are
uniform and consistent, both within and between preps. Nextera
XT has cut down on our library preparation headaches, and ourexperience with the kit has been extremely positive.

“With Nextera XT, we consistently get
20 million reads and upwards of 5 billion
base pairs from a single MiSeq run.”
Q: What applications have you developed?
TS: We’ve focused our applications on bacterial genomes because
a number of our investigators are interested in virulent and diseaserelated microbes. It is a rapidly emerging area. Thanks toNextera XT, we’ve been able to process more microbiome studies.
Microbial genome sequencing is relatively straightforward for us now.
We’ve also started using the Nextera Mate Pair Library Preparation
kit with both the gel-free, and the gel-plus protocols, and havebeen successful in assembling complete, single-chromosome
genomes. Having larger mate pair libraries has greatly facilitated theassembly of our genomes. The Illumina approach is cost-effective
for whole-genome sequencing, and the Nextera Mate Pair gel-plus
protocol has made a significant difference for us in applications
requiring a specific mate pair size. We’re amazed at what we’vebeen able to accomplish in less than a year.
Q: How has Nextera XT improved your workflow?
TS: In terms of processing time, you can’t beat the turnaroundtime of Nextera XT. Instead of taking one or two days, we canproduce 96 libraries in less than a day, and load the MiSeq flow
cell at the end of the day. We’re a small core facility so our volumes
aren’t necessarily rate-limiting for us. Nextera XT provides moretime for researchers who produce the libraries to work on otherapplications. Nextera XT allows our investigators to spend less timemaking libraries and more time making discoveries.

Q: How have the Nextera XT kits improved your data results?
TS: We typically sequence 10 microbial genomes at a time. With
the Nextera XT kit, we can process multiple samples up to 96 quiteeasily. One of our evolutionary biologists is interested in beetles,
which have 200–300 million base pair genomes. We were able
to generate a complete sequence of an insect for him using theMiSeq System and Nextera XT. The increase in read length and thenumber of reads make that possible. We consistently get 20 million
reads and upwards of 5 billion base pairs from a single MiSeq run.
That’s amazing when you think about it.
Q: How do you measure the quality of the sequence data?
TS: The Q30 value is important to us and we’ve obtained longread lengths with high Q values using the Nextera XT kit. Generally,
we get one error in 1,000 base pairs, which is consistent acrossmost of the read length, especially on the first read. We also lookat the distribution of the library. When we sequence genomes
that have a reference genome, we simply align our reads tothat reference sequence. Bacterial genomes can be difficult tosequence due to high nucleotide repeat regions. Because theNextera XT fragmentation process reduces enzymatic bias in repeat
regions, we get better library diversity, producing a distributionthat randomly covers the genome without introducing sequenceor technology bias. Our initial alignments and quality of data havegiven us high confidence in the Illumina technology.
Q: What applications do you plan to introduce next?
TS: We’d like to move away from smaller amplicons so we’reinterested in optimizing long-range PCR. Coupled with theefficiency of Nextera XT, long-range PCR would be an ideal
application for resequencing projects and studies involving
repetitive sequencing for taxonomy or evolutionary purposes.
We’ve already worked out the Nextera XT protocol and theamplicon sequencing is coming along nicely.
Q: What advice do you have for researchers considering
Nextera XT Kits?
TS: Each application brings its own nuances. The Illumina FAS
team is really adept at helping to troubleshoot those. After you
learn the basics, the rest comes easily, and an Illumina FAS isthere to provide training at every step. The Nextera XT kit has beenparticularly useful for sequencing our microbial genomes and has
been a great experience for us. I highly recommend Nextera XT formicrobiome studies.
To learn more about the MiSeq Desktop Sequencer, visit
www.illumina.com/miseq.
For more information on the Nextera XT DNA Library Preparation
kits, visit www.illumina.com/products/nextera_xt_dna_sample_prep_kit.ilmn.

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Pub. No. 770-2013-040 Current as of 19 November 2014