October 2014

Sifting Through Cells
Advances in sequencing technology like the NextSeq® 500 system are giving scientists a peekinto the processes that guide cellular development.

Introduction
A single cell in the human body boasts about 6 billion basepairs of DNA and 600 million bases of RNA. Understanding
how this deluge of information works to construct healthyorgans or how it breaks down to provoke cancer, istricky. Biologists must meticulously comb through thesesequences to grasp thoroughly how these molecular
instructions make cells tick.
At Stanford University Medical Center, DNA Sequencing
Core Director Norma Neff, Ph.D., manages support for ateam that is sequencing RNA with the NextSeq 500 system
to parse the thousands of genes that assemble vital organs
such as the brain and pancreas. The team is led by Stephen
Quake, D. Phil., a genomics innovator and Professor of
Bioengineering at Stanford. Instead of exploring the genetic
makeup of cell populations, the team uses a techniquecalled single-cell sequencing, which reads the gene
expression of one individual cell at a time. In this way, thegroup can catch the unique genetic composition of a cellof interest, rather than relying on the consensus in a groupof cells. This effort is part of a grant from the California
Institute for Regenerative Medicine (CIRM).
Before Dr. Neff was recruited to be director of theSequencing Core at Stanford by Dr. Quake in 2008, herwork in yeast genetics at Memorial Sloane Kettering Cancer
Center in New York helped discover a yeast protein withan intein that is capable of splicing itself. She also workedon the rare inherited disorder called Bloom syndrome,
which makes a person more cancer-prone, at the New York
Blood Center.
iCommunity spoke with Dr. Neff to understand how singlecell sequencing is transforming our understanding of thedevelopmental programs that drive cells toward healthand disease.
Q: When did you start using Illumina sequencing systems?
Norma Neff (NN): I started working with Illumina sequencing
systems with the ENCODE (Encyclopedia of DNA Elements)
project in 2007 with Richard Myers, Chairman of Genetics
at Stanford. We were looking at binding sites for different
transcription factors. When I started working with Steve
Quake, the Stem Cell Institute had moved off campus to abuilding on Arastradero in Palo Alto. I was recruited to help set
up the sequencing core for the Stem Cell Institute in 2008.

Photo courtesy of Jun Seita

The Stanford DNA Sequencing Core is located in the Lokey Stem Cell building,where a Dale Chilhuly glass chandelier graces the lobby.

Q: The team at Stanford has a grant from CIRM to perform
single-cell sequencing on the brain and the pancreas. What
are the goals of your studies?
NN: We’re really digging into these organs to understand theirdevelopment and characterize the different cell types in a
precise way. We’re trying to develop gene sets that will identify
specific cell types in these various organs. The idea is to see
what we can learn from the most abundant and moderately
expressed genes in the cell. With single-cell RNA-Seq (mRNASequencing), you can look specifically at gene expression
patterns within individual cells.
Q: Does this work have applications in cancer?
NN: Prospectively, down the road we’ll work on cancers, butright now the basic focus is on early development. Recent
studies suggest that cancers arise in a stem or early progenitor
cell, so a deeper understanding of the normal moleculardevelopmental program will help us understand how cells
become tumorigenic.
Q: Why is this work so challenging?
NN: Single, isolated cells are fragile and contain very small
amounts of mRNA. Contamination and RNA degradation are
constant problems.
Q: What observations have you made?
NN: We’ve learned that there aren’t as many genes expressedin single cells as originally thought. When looking at theENCODE project, there are 15,000 to 20,000 genes expressedin the cell populations. With single cells, we really getsomewhere between 6,000 and 8,000 genes being expressedOctober 2014
in individual cells. So you don’t have to sequence as deeply as
you do for other types of RNA-Seq. Also there are disparities
between mRNA levels and protein expression. Single cells thatare sorted using antibodies to abundant surface markers don’t
always have abundant mRNA for that protein.
Q: Why did you choose the NextSeq 500 system?
NN: We chose it for the high number of reads you get andthe fast turnaround. The NextSeq 500 System has really been
the backbone of this pipeline, enabling us to pool 100–300individual single-cell RNA-Seq libraries together and sequencethem. We’ve been using the new 384-index system for theNextera® libraries and that’s helped out a lot. If we find cells
that look more interesting and want to sequence more deeply
using the large index set, the probability that you can go backand sequence that one cell more deeply in another pool ismuch higher with more indexes.
Q: How has the NextSeq 500 system performed?
NN: We’ve been running it quite successfully six days a week;
we give it one day off. Primarily, we perform 2 x 75 bp runs
for the RNA-Seq libraries. That’s a perfectly good read length
when you’re just aligning it to the reference genome. It keeps
the error rate low, everyone is very happy with the data quality,
and the cost per Gb is nearly identical to HiSeq® system runs.

“The data quality that we getoff the NextSeq 500 system at
2 × 75 bp is exactly what we’relooking for in our studies.”
Q: How does the NextSeq 500 system compare to theMiSeq® system you have in your lab?
NN: The data quality that we get off the NextSeq 500 system
at 2 × 75 bp is exactly what we’re looking for in our studies.
The flow cells are shipped dry so there is no cleaning. Aftersequencing, the system automatically washes itself using a
bleach solution added into the reagent cassette, so there isno delay in starting the next run. From a speed standpoint, it
actually makes the MiSeq® system look slow.
Q: What library prep kit are you using?
NN: We’re using the Illumina Nextera XT® kit very successfully.
We’re really glad to have it, primarily because we makehundreds of libraries at a time. The 96 capillary AATI Fragment
Analyzer is a critical instrument in our single-cell sequencing
pipeline. Once you quantitate the cDNA (complementaryDNA) with capillary electrophoresis, the Nextera libraries are
so reproducible that you just mix 2 microliters of your libraries
and clean up a single pool and submit them for sequencing.
There’s no need to quantitate the individual library preps

because they’re coming out to be so uniform from plate toplate. We’re very happy with this product.

“It’s both the throughput and theturnaround time of the NextSeq
system that is helping to advance
this research field.”
Q: What types of sequencing studies are you performingwith the MiSeq system?
NN: We now use the MiSeq system for experimental library
construction and for metagenomics primarily. We’re doing alot of metagenomics in collaboration with JGI (Joint GenomeInstitute). The long-term goal of the project is to develop
methods for sequencing and assembling single cell bacterial
genomes from environmental samples. We also used theMiSeq system to sequence single nucleotide polymorphisms(SNPs) for cell lineage studies from various types ofcancer cells.
Q: Why is now the time for single-cell sequencing?
NN: The technology to isolate cells and amplify RNA has
gotten much better. We rely on the Fluidigm C1 system
to capture single cells and synthesize cDNA in nanoliter
microfluidic chambers. There’s an opportunity for people to
make an unbiased assessment of the cells in their population
with regard to mRNA expression.
Illumina sequencing has improved to the point where theturnaround time is much faster and the throughput is highenough to be able to get 1–2 million reads for several
hundred single cell libraries in a 30-hour run. It’s quick
enough to support multiple single-cell projects with timely
data production. It’s both the throughput and the turnaroundtime of the NextSeq system that is helping to advance thisresearch field.
Q: What improvements must be made to make single-cellsequencing more useful or universally available?
NN: We are focused on improving protocols for making single
cell suspensions and investigating novel ways of displaying
the data. Making single suspensions out of solid tissue is aparticular challenge, primarily because you have this inherent
feeling that the gene expression is changing at the time you’redoing your prep. So the sooner the cells can be captured
and made into cDNA, the better off you’ll be. Collecting,analyzing, and displaying the data in useful ways are all part ofan evolving process. Integrating and sharing this new data inan efficient and friendly format among different institutions is along-term goal of the CIRM project.

October 2014
Q: What are the next trends in this field?
NN: The Stem Cell Institute is interested in defining what are
the true stem cells, both for various types of developmental
programs as well as cancer stem cells. We’re trying to isolate
and transplant these cells to see what their potential is andlearn something about their gene expression program.
We collaborate with researchers in the Stem Cell and Cancer
Institutes to try to find new cell surface markers and newtranscription factors by single-cell RNA-Seq to help develop
better tools to isolate these cells in a purer form, especiallywith regard to stem cells and cell-based therapy. You want togive people the right cells.
For cancer therapy to focus on cancer stem cells, you want tobe able to target the correct cell population without causing
damage to other cells. That’s not really a new trend, but
the technology has gotten better. By looking at cells on an
individual basis we have a new window to try to understandwhat these cells are, what they’re doing, and how to target
them more effectively.

October 2014Illumina • 1.800.809.4566 toll-free (U.S.) • +1.858.202.4566 tel • techsupport@illumina.com • www.illumina.com
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affiliate(s) in the U.S. and/or other countries. Pub. No. 770-2014-015 Current as of 30 October 2014