ILLUMINA® SEQUENCING

Multiplexed Sequencing with the

Illumina Genome Analyzer System
Introducing index sequences onto DNA fragments enables sequencing of 96 different samples
on a single flow cell. This greatly increases experimental scalability, while maintaining extremely
low error rates and conserving read length.

HIGH-THROUGHPUT SEQUENCING
Using the industry’s leading next- FIGURE 1: MULTIPLEXED SEQUENCING PROCESS

generation sequencing technology, A. READ 1 B. INDEX READ C. READ 2

the Genome Analyzer system offers

proven, exceptionally high data
yields and the largest number of Rd1 SP
error-free reads. Harnessing this se- Rd2 SP

quencing power in a multiplex fash-

ion increases experimental through-
put while reducing time and cost. DNA DNA
insert insert
This is especially useful when target-
ing genomic sub-regions or studying Index SP
small genomes. To make multiplexed
Index
sequencing on the Genome Analyzer
available to any laboratory, Illumina
offers the Multiplexing Sample
Preparation Oligonucleotide Kit and
the Multiplexing Sequencing Primers Sample multiplexing involves a total of three sequencing reads, including a separate
index read, which is generated automatically on the Genome Analyzer equipped with
and PhiX Control Kit. the Paired-End Module. A: Application read 1 (dotted line) is generated using the
Read 1 Sequencing Primer (Rd1 SP). B: The read 1 product is removed and the Index
Sequencing Primer (Index SP) is annealed to the same strand to produce the 6-bp in-
dex read (dotted line). C: If a paired-end read is required, the original template strand
HIGHLIGHTS OF ILLUMINA
MULTIPLEXED SEQUENCING is used to regenerate the complementary strand. Then, the original strand is removed
and the complementary strand acts as a template for application read 2 (dotted
line), primed by the Read 2 Sequencing Primer (Rd2 SP). Pipeline Analysis software
• Fast, High-Throughput
identifies the index sequence from each cluster so that the application reads can be
Strategy: Automated sequencing
assigned to a single sample. Hatch marks represent the flow cell surface.
of 96 samples per flow cell

• Cost-Effective Method: Multi-

sample pooling improves In the multiplexed sequencing for individual downstream analysis.
productivity by reducing time method, DNA libraries are “tagged” Using this approach, sample
and reagent use
with a unique identifier, or index, identification is highly accurate.
• High-Quality Data: Accurate during sample preparation. Multiple
maintenance of read length for APPLICATIONS
samples are then pooled into a single
unknown sequences Multiplexed sequencing on the
lane on a flow cell and sequenced
Genome Analyzer can be used in
• Simplified Analysis: Automated together in one Genome Analyzer
a wide range of applications. For
sample association with index run. An automated three-read
using Pipeline Analysis software example, following genome-wide
sequencing strategy (Figure 1) identi-
association studies of human
fies each uniquely tagged sample
 ILLUMINA® SEQUENCING

disease, multiplexed sequencing can fragments, avoiding constraints on performed in parallel with novel
be performed for time- and cost- read length or fragment size and fluorescently labeled reversible ter-
effective resequencing of targeted maximizing yield and data utility. minator nucleotides. A total of three
regions in many individuals. Multi- Prepared samples can be used on sequencing reads are performed
plexed sequencing can also be used both single-read and paired-end (Figure 1). The first read is identical to
to characterize small, non- flow cells. that of a paired-end experiment and
human genomes, such as when uses the standard Read 1 Sequencing
determining genetic variations FULLY AUTOMATED SEQUENCING Primer provided in the Paired-End
between bacterial strains responsible The multiplexed sequencing process Cluster Generation Kit. At the end of
for separate disease outbreaks. is fully automated using the Genome the first read, the extended sequenc-
In studies of gene structure and Analyzer, Cluster Station, and Paired- ing primer is removed and the Index
regulation, multiplexed sequencing End Module. Sequencing Primer, provided in the
can be applied to whole-transcrip- The Cluster Station amplifies DNA Multiplexing Sequencing Primers and
tome sequencing and to DNA from up to 96 samples on the flow PhiX Control Kit, is annealed to the
recovered by chromatin immunopre- cell surface to create clusters same strand. This approach lever-
cipitation (ChIP) experiments. containing 500–1,000 clonal copies of ages the Paired-End Module to avoid
each molecule. The resulting the loss of high-quality sequencing
UNIQUE INDEX TAGS high-density array of templates is data from the unknown sample that
In a multiplexed run on the Genome sequenced using the Genome would occur if the index sequence
Analyzer, multiple samples are Analyzer and the Paired-End Module. had been included at the start of an
sequenced in a single lane of a flow Sequencing by synthesis is application read.
cell. To identify samples after pool-
ing, each sample is uniquely tagged
with a sequence index during the FIGURE 2: THE MULTIPLEXED SEQUENCING SAMPLE PREPARATION METHOD
FOLLOWS THE FAMILIAR PAIRED-END PROTOCOL
sample preparation protocol.
The Multiplexing Sample Prepara-
tion Oligonucleotide Kit provides Purified DNA fragments (< 800 bp)
12 index oligos for pooling up to
Repair ends
12 samples per lane, or 96 samples
Blunt-ended fragments with
per flow cell. Each index is six bases
5'-phosphorylated ends
in length. This permits accurate
Add an ‘A’ to the 3’ ends
differentiation between samples, even
if an index read contains an error. 3’-dA overhang

SIMPLE SAMPLE PREPARATION Ligate novel paired-end adapter

Sample preparation for multiplexing
Adapter-modified ends
on the Genome Analyzer is highly
robust and familiar. The straightfor- Purify ligation product
ward workflow requires as little as Removal of unligated
one microgram of input DNA. It is adapters
based on the simplified Paired-End PCR using two novel universal
primers plus index primer
sample preparation procedure with
minimal changes (Figure 2). Index Indexed DNA library

sequences are added to adapter-

modified DNA fragments during the
The multiplexed sequencing sample preparation method follows standard sample
PCR enrichment step (Figure 3). The preparation protocols for paired-end sequencing, with the exception of using a
protocol does not require use novel adapter and PCR primers (shown in red).
of restriction enzymes to prepare
 ILLUMINA® SEQUENCING

Prior to application read 2, the

index sequencing product is removed FIGURE 3: ADDING THE SEQUENCE INDEX TO A LIBRARY
and the clusters are modified in situ
to regenerate the complementary
Rd1 SP DNA Insert
strand using the Paired-End Module.
The Multiplexing Read 2 Sequencing
Primer is annealed to the comple-
1. During sample preparation, adapters are ligated to the DNA fragments.
mentary strand and extended to One adapter contains the sequencing primer site for application read 1
complete the final read. (Rd1 SP).

Using Illumina’s Pipeline Analysis

software, each index is associated
with a particular read-pair, identify-
ing samples for downstream analysis.

HIGH-QUALITY DATA
Sample multiplexing on the Genome
Analyzer system produces high-
throughput sequence information
P5 Index SP
with industry-leading accuracy.
Data quality is equivalent to that Rd2 SP
routinely achieved in a single-read
2. Prepared samples are amplified via PCR using two universal primers. One
or paired-end run. Due to the inher- primer contains an attachment site (P5) for the flow cell, while the other
ent redundancy in the index design, contains the sequencing primer sites for the index read (Index SP) and
for application read 2 (Rd2 SP).
both perfect index reads and those
that differ by one base can be used
as sample identifiers (Figure 4).
Index
AUTOMATIC SAMPLE IDENTIFICATION P7
AND PROCESSING
Pipeline Analysis software (version 1.0
3. A third primer in the PCR adds the Index as well as a second flow cell
and higher) includes the ability to attachment site (P7) to the PCR product shown in step 2.
discriminate between the three
reads. Once the sequencing chemistry
is complete, the alignment software
P5 Rd1 SP DNA Insert Index SP Index P7
identifies the index sequence and
annotates each read with its
Rd2 SP
respective index. From this point
4. The indexed library is ready for sequencing using the Genome Analyzer
on, reads derived from a given
system.
multiplexed sample can be positively
identified.
Just as in a non-multiplexed
detecting structural variation, and UNLIMITED ACCESSIBILITY
sequencing run, Pipeline software
assembling sequences de novo. The Multiplexing Sample Preparation
provides automated base-calling,
Like all of Illumina’s software Oligonucleotide Kit and Multiplexing
calculation of quality values for
solutions, Pipeline software offers Sequencing Primers and PhiX Control
every base, and alignment of read
an open architecture that allows for Kit simplify high-throughput
pairs to a reference sequence. The
easy customization of downstream multiplexed sequencing with the
ability to combine multiplexing
analysis and integration with a vari- Genome Analyzer system. In addition
with paired-end reads is crucial
ety of innovative analysis tools. to multiplexing, the Genome
for optimizing sequence alignment,
 ILLUMINA® SEQUENCING

Analyzer system can be used for

FIGURE 4: PERCENTAGE OF USABLE INDEX READS single-read sequencing, mRNA-Seq,
ChIP-Seq studies, and more. As an
100
open platform for genetic analysis,
the Genome Analyzer enables the

80
broadest range of applications. View
Percent of Usable Index Reads

the latest accomplishments using

Illumina sequencing technology at
60 www.illumina.com/publications.

40

20

0
1 2 3 4 5 6 7 8 9 10 11 12
Library Number

Perfect index reads Single-error index reads

Most index sequences are perfect, but error correction in the index design means
that even the small number of reads with a single error can be used. Twelve libraries
were prepared, each tagged with a different index, and sequenced in individual flow
cell lanes. The percentage of index sequences that can be used (bar height) is a com-
bination of perfect index reads (blue) and those with a single error (grey).

ORDERING INFORMATION

PRODUCT DESCRIPTION CATALOG NO.

Multiplexing Sample Preparation Kitted oligonucleotides used to prepare up to PE-400-1001

Oligonucleotide Kit 96 samples for multiplexed sequencing*.

Multiplexing Sequencing Primers Kitted multiplexing sequencing primers, multiplexing PE-400-1002

and PhiX Control Kit control DNA, and buffer set. Sufficient for up to
10 Genome Analyzer runs.

*Requires Genomic DNA or Paired-End Sample Prep Kits, available separately.

ADDITIONAL INFORMATION Illumina, Inc.

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techsupport@illumina.com
www.illumina.com

FOR RESEARCH USE ONLY

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Pub. No. 770-2008-011 Current as of 2 December 2008