June 2015

Unraveling Disease Mechanisms With The
NextSeq 500 System®

Researchers at the Icahn School of Medicine at Mount Sinai are studying mtDNA, piRNA, andTCR repertoire diversity to unlock the secrets of cancer, infertility, and autoimmune disease.
Introduction
Extensive research is focused on how human genome aberrations
contribute to the onset and progression of disease. Structuralvariations, gene mutations, deletions, and insertions have all beenfound to play a role. Yet up until recently, researchers have been
limited in studying how genetic changes in the DNA or RNA driving
specific biological subsystems might be involved in disease onset
and progression.
An innovative research team at the Icahn School of Medicine at Mount
Sinai led by Ravi Sachidanandam, PhD, is focused on developing newmethods to study 3 of these important subsystems: mitochondrial
DNA (mtDNA), T cell receptor (TCR) diversity, and the piwi-interacting
RNA (piRNA) pathway. Each has been difficult to study in the past.
mtDNA studies are challenging, because it is hard to parse out
mtDNA from the whole genome for sequencing purposes to identify itspotential role in disease processes. Sequencing the full TCR repertoireis time-consuming, limiting detailed study of the adaptive immune
system, its role in autoimmune disease, and immune and vaccine
response. Researchers have also faced challenges in deciphering roleof the piRNA pathway in defending the germline against attacks fromtransposons that could impact fertility.
Working with Illumina sequencing systems for the past 5 years,Dr. Sachidanandam and his team invented a new method for removing
biases in small RNA sequencing while studying piRNAs1. They also
developed a method to sequence mtDNA with high resolution,
without having to simultaneously sequence the nuclear genome2, andare working on new, sequencing-based approaches for monitoring
TCR response in blood. In 2015, his team installed a NextSeq 500
System in the Oncological Sciences department to expedite theirresearch studies.

The chair of genetics at Mount Sinai Hospital recruited me 5 years ago
to build upon the strictly computational work that I had been doingat Cold Spring Harbor. When I made the move, they were thinkingof starting a sequencing program and the responsibility for setting it
up fell into my hands. I hired a technician and together we learned
a lot about sequencing systems and workflows as we establishedthe laboratory.
Q: How are your team’s research goals intertwined with thedevelopment of new sequencing methodologies?
RS: It becomes a means to an end. We are investigating dynamic
biological subsystems that researchers have struggled to analyze.
We find that there is a continual loop where the computational resultsof our RNA and DNA sequencing efforts inform our wet-bench
practices and vice versa. This flow of information has enabled us todevelop innovative and powerful approaches, including new laboratory
techniques, and computational algorithms and tools.
Our piRNA research is a good example, because it led to our firstsequencing invention. piRNAs are the largest class of small, noncoding
RNA molecules and play a role in gene silencing, specifically thesilencing of transposons. They are present only in the germline.
Alterations in the piRNA pathway can disrupt protection of thegermline from transposon attack, potentially resulting in infertility.

iCommunity spoke with Dr. Sachidanandam to learn how his team isusing the NextSeq 500 System to develop novel sequencing methods.
Q: What sparked your interest in genomics?
Ravi Sachidanandam (RS): I was trained as a physicist and as I was
thinking of what to do after my postdoc, I was reading a lot about
genomics in newspapers and journals. I applied to the Cold Spring
Harbor Laboratory and began working on a large-scale SNP project,which was the very first of its kind, to compile SNPs in the humangenome. The database included > 300,000 SNPs. I eventually ended
up working on several projects, which included a group that wereamongst the first to generate data on the early Illumina sequencing
systems, the Genome Analyzer® II.

For Research Use Only. Not for use in diagnostic procedures.

Ravi Sachidanandam, PhD, is an Assistant Professor of Oncological
Sciences at the Icahn School of Medicine at Mount Sinai.

June 2015
We accidentally discovered that the protocols used for small RNA
sequencing are biased towards certain piRNA. We were able to inventa new method of sequencing small RNAs. The method works well forpiRNAs and other small RNAs, including miRNAs.
Q: What other scientific questions are you trying to answer?
RS: There are 2 other biological subsystems that we are studying andtrying to understand in greater depth. One of them is the genetics of
mtDNA. Mitochondria are the engines of our cells, converting foodinto energy to fuel our muscles, brain, and other tissues. Sequencing
mtDNA is difficult. In the past, we sequenced the whole genome and,in the process, picked up some mtDNA pieces. So while the role ofmitochondria in disease might be widespread, we have not been able
to decipher it because of our inability to sequence mtDNA and thegenome separately.
The other subsystem we are studying is TCRs, which are part of ouradaptive immune system. Each person’s TCR repertoire possesses
unique elements that are triggered by its response to viral and bacterial
infections over their lifetime. It takes over when our immune system
mistakes our own cells for a foreign entity and starts attacking them.
TCRs also play a role in vaccine responses and how quickly the bodyclears an infection. We are developing new sequencing approaches totrack these responses.

“We find that there is a continual
loop where the computational
results of our RNA and DNA
sequencing efforts inform ourwet-bench practices and viceversa. This flow of information has
enabled us to develop innovativeand powerful approaches,
including new laboratory
techniques, and computationalalgorithms and tools.”

DNA through an enzymatic process, leaving behind the circular
mtDNA. From that point on, it is a simple process to sequence themtDNA at very high resolution, about 1000× coverage.
Using this unique method, we screened large numbers of humancell lines and discovered the mtDNA diversity is widespread andstable. Since then, we have developed an entire program focusedon understanding how the mitochondrial genome affects humandevelopment and metabolic disorders.
Now there is kind of a “gold rush” in this field because we can actuallymonitor and track mitochondria in different tissues using this techniqueand understand what is going on in various disorders.

“The biggest advantages of theNextSeq 500 System are its
throughput, optimal price point,
and fast turnaround time. The
installation was easy and we wereable to start running projects on
it immediately.”
Q: Can you talk about the TCR technique that you are developing?
RS: We have developed a method of sequencing to monitor the TCR
repertoire in people over time, potentially using it as a biomarker for
disease. The key point is the use of universal primers for unbiased
sampling of the repertoire, avoiding the biases that plague otherapproaches that are mostly based on PCR amplification. Using thetechnique we could learn how people react to different diseases orwhether a vaccination is working. There are also potential implications
for cancer immunogenomics.
Q: On which diseases is your research focusing?
RS: I am in the Oncological Sciences department, so clearly cancer
is a focus. Within mtDNA, we are looking at what goes on in tumors
and if there is an evolution of the mitochondrial genome as the tumor
progresses. I also conduct research in autoimmune disease andcollaborate with Dr. Terry Davies, a famous researcher of Graves’disease, an autoimmune thyroid disorder. We are trying to understandthe role of the TCR repertoire in this disorder.

Q: Can you talk about the Mseek technique that yourteam developed?
RS: The Mseek technique solved the mtDNA sequencing problem. In
the past, when we sequenced cellular DNA, only a small amount of it
was mtDNA and our resolution was poor. That is not surprising. The
nuclear genome consists of 6 billion bases, while the mitochondrial
genome is only 17,000 bases. Even though there are 1,000 mtDNA
for every nuclear genome, total mtDNA adds up to only 17–20 million
bases. So less than 1% of total DNA is mtDNA.

Q: Does your research support personalized medicine approaches
to fighting disease?
RS: That is the goal, particularly of our TCR research. The idea is tobe able to monitor disease through noninvasive techniques. Ultimately,we would like to be able to take a small blood sample and use ourapproach to figure out what the immune system is doing. It wouldenable us to tailor therapies to each person.

Anitha Jayaprakash, a student in the lab, invented Mseek, a methodof isolating and sequencing just the mitochondrial DNA. The techniquetakes advantage of the fact that the mitochondrial genome is circularand the nuclear genome is linear. Mseek eliminates the linear nuclear

Q: How important are the tools that you use to the success of
your research?
RS: The technologies and tools we use are extremely important.
Sequencing is our main technique because we use it to infer thingsfrom tissue and fluid samples. Access to technology and a quick

For Research Use Only. Not for use in diagnostic procedures.

June 2015
data turnaround are important for us because we develop techniques
to address specific areas. For example, if I want to look at the TCR
repertoire, I have to develop a method to monitor it in the blood. Wespend a lot of time going back and forth—taking a sample, using amethod to prepare and sequence it, looking at the sequencing data,
and tweaking the method until we are sure that everything is working.
Then we apply it to large sets of samples.
Q: How long have you been using the Illumina systems?
RS: I have been using Illumina systems since the Genome Analyzer
II System was introduced in 2006. In this lab, we have used almost
every Illumina sequencing system, including the MiSeq®, HiSeq®, andNextSeq Systems.

bottleneck. Previously we would wait up to 8 weeks for data; now we
get results back in a day. Data turnaround is not really a considerationin planning experiments anymore. Our work volume has increased,enabling us to pursue additional research directions.
Q: How many different sequencing applications are you performingwith it?
RS: We perform mtDNA sequencing and the TCR repertoire analysis
using our proprietary techniques. We are also performing mRNA-Seq,
ChIP-Seq, whole-exome sequencing, amplicon sequencing, and somebacterial genome sequencing.

“Previously we would wait
up to 8 weeks for data; now
we get results back in a day.
Data turnaround is not reallya consideration in planning
experiments anymore.”

Q: How do the Illumina systems compare with othersequencing systems?
RS: The systems are good in terms of ease of use. The
responsiveness of the Illumina technical support team has been
important for us. We might be one of the heaviest users of thisservice because we are always trying pushing the boundaries ofthe sequencer, trying new things, wanting to customize this or that.
Illumina Tech Support always tries to address our questions and help
us find ways to perform what we are trying to do. That is a significant
plus for Illumina, and as a customer I appreciate the added dedication.

“Having our own NextSeq 500
System has let us generate
data and get feedback in thelab quickly. It has eliminated oursequencing bottleneck.”
Q: Why did you choose the NextSeq 500 System for yourcurrent study?
RS: The biggest advantages of the NextSeq System are its
throughput, optimal price point, and fast turnaround time. The
installation was easy and we were able to start running projects on
it immediately.
The NextSeq System enables us to have real-time monitoring of
our experiments so we can go back and forth between the lab,
analysis, and sample preparation. We can quickly solve problems in
the lab before we get too far. Before, we had to wait a few weeks fordata and would lose track of what was going on in the lab. After 3
months working with the NextSeq 500 System, we have significantly
shortened our debug cycle for the techniques we are developing.
Q: What do you think of its performance and data quality?
RS: Our yields exceeded the specified output significantly and thedata quality has been comparable to what we used to get from theHiSeq System.
Before installing the NextSeq System, we were using a HiSeq
System at a core lab and at various companies, or a MiSeq System.
Having our own NextSeq 500 System has let us generate data andget feedback in the lab quickly. It has eliminated our sequencing

For Research Use Only. Not for use in diagnostic procedures.

Q: Do you use any Illumina library prep kits?
RS: For mRNA-Seq, we use the TruSeq® Stranded mRNA Library
Prep Kit.
Q: What types of bioinformatics tools have you developed?
RS: We have developed all the bioinformatics tools we use in-house.
We have several tools, resources, databases, and websites that allow
others to analyze the data on our website. For example, Kismeth
is a web-based tool for bisulfite sequencing analysis that enables
researchers to analyze methylation states in plants and humans. OurmRNA-splicing resource, SpliceRack, is widely used in the field as are
our tools for piRNA analyses.
Q: What are the next steps in your research?
RS: We will apply our tools to various problems. For example, we will
be using the Mseek protocol to study mitochondrial genetics in a twins
study, comparing mitochondrial genomes and seeing how they differ.
Since twins come from the same mother, they should technically haveidentical mitochondrial genomes. We are testing that theory. We also
want to compare the TCR repertoire in twins and see how they diverge
over time.

References:1.

2.

Jayaprakash AD, Jabado O, Brown BD, and Sachidanandam R.
Identification and remediation of biases in the activity of RNA ligases insmall-RNA deep sequencing. Nucleic Acids Res. 2011; 39(21):e141. doi:
10.1093/nar/gkr693.
Jayaprakash AD, Benson EK, Gone S, et al. Stable heteroplasmy at thesingle-cell level is facilitated by intercellular exchange of mtDNA. Nucleic
Acids Res 2015; 43(4): 2177–2187.

June 2015

Learn more about the Illumina systems andproducts mentioned in this article:••••NextSeq 500 System, www.illumina.com/systems/nextseqsequencer.html
MiSeq System, www.illumina.com/systems/miseq.html
HiSeq System, www.illumina.com/systems/hiseq_2500_1500.html
TruSeq Stranded mRNA Library Prep Kit,
www.illumina.com/products/truseq_stranded_mrna_library_prep_kit.html

Illumina • 1.800.809.4566 toll-free (US) • +1.858.202.4566 tel • techsupport@illumina.com • www.illumina.com
For Research Use Only. Not for use in diagnostic procedures.
© 2015 Illumina, Inc. All rights reserved. Illumina, Genome Analyzer, HiSeq, MiSeq, NextSeq, TruSeq, and the pumpkin orange color are
trademarks of Illumina, Inc. and/or its affiliate(s) in the U.S. and/or other countries. Pub. No. 770-2015-017 Current as of 23 June 2015