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 fl ow 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-
`generation sequencing technology,
`the Genome Analyzer system offers
`proven, exceptionally high data
`yields and the largest number of
`error-free reads. Harnessing this se-
`quencing power in a multiplex fash-
`ion increases experimental through-
`put while reducing time and cost.
`This is especially useful when target-
`ing genomic sub-regions or studying
`small genomes. To make multiplexed
`sequencing on the Genome Analyzer
`available to any laboratory, Illumina
`offers the Multiplexing Sample
`Preparation Oligonucleotide Kit and
`the Multiplexing Sequencing Primers
`and PhiX Control Kit.
`
`HIGHLIGHTS OF ILLUMINA
`MULTIPLEXED SEQUENCING
`
`(cid:129)
`
`(cid:129)
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`(cid:129)
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`
`Fast, High-Throughput
`Strategy: Automated sequencing
`of 96 samples per fl ow cell
`
`Multi-
`Cost-Effective Method:
`sample pooling improves
`productivity by reducing time
`and reagent use
`
`Accurate
`High-Quality Data:
`maintenance of read length for
`unknown sequences
`
` Automated
`Simplifi ed Analysis:
`sample association with index
`using Pipeline Analysis software
`
`FIGURE 1: MULTIPLEXED SEQUENCING PROCESS
`
`A. READ 1
`
`B. INDEX READ
`
`C. READ 2
`
`Rd1 SP
`
`DNA
`insert
`
`Rd2 SP
`
`DNA
`insert
`
`Index SP
`
`Index
`
`Sample multiplexing involves a total of three sequencing reads, including a separate
`index read, which is generated automatically on the Genome Analyzer equipped with
`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
`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
`identifies the index sequence from each cluster so that the application reads can be
`assigned to a single sample. Hatch marks represent the flow cell surface.
`
`In the multiplexed sequencing
`
`method, DNA libraries are “tagged”
`with a unique identifi er, or index,
`during sample preparation. Multiple
`samples are then pooled into a single
`lane on a fl ow cell and sequenced
`together in one Genome Analyzer
`run. An automated three-read
`sequencing strategy (Figure 1) identi-
`fi es each uniquely tagged sample
`
`for individual downstream analysis.
`Using this approach, sample
`identifi cation is highly accurate.
`
`APPLICATIONS
`Multiplexed sequencing on the
`Genome Analyzer can be used in
`a wide range of applications. For
`example, following genome-wide
`association studies of human
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`Ariosa Exhibit 1010, pg. 1
`IPR2013-00276
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`ILLUMINA® SEQUENCING
`
`disease, multiplexed sequencing can
`be performed for time- and cost-
`effective resequencing of targeted
`regions in many individuals. Multi-
`plexed sequencing can also be used
`to characterize small, non-
`human genomes, such as when
`determining genetic variations
`between bacterial strains responsible
`for separate disease outbreaks.
`In studies of gene structure and
`regulation, multiplexed sequencing
`can be applied to whole-transcrip-
`tome sequencing and to DNA
`recovered by chromatin immunopre-
`cipitation (ChIP) experiments.
`
`UNIQUE INDEX TAGS
`In a multiplexed run on the Genome
`Analyzer, multiple samples are
`sequenced in a single lane of a fl ow
`cell. To identify samples after pool-
`ing, each sample is uniquely tagged
`with a sequence index during the
`sample preparation protocol.
` The Multiplexing Sample Prepara-
`tion Oligonucleotide Kit provides
`12 index oligos for pooling up to
`12 samples per lane, or 96 samples
`per fl ow cell. Each index is six bases
`in length. This permits accurate
`differentiation between samples, even
`if an index read contains an error.
`
`SIMPLE SAMPLE PREPARATION
`Sample preparation for multiplexing
`on the Genome Analyzer is highly
`robust and familiar. The straightfor-
`ward workfl ow requires as little as
`one microgram of input DNA. It is
`based on the simplifi ed Paired-End
`sample preparation procedure with
`minimal changes (Figure 2). Index
`sequences are added to adapter-
`modifi ed DNA fragments during the
`PCR enrichment step (Figure 3). The
`protocol does not require use
`of restriction enzymes to prepare
`
`fragments, avoiding constraints on
`read length or fragment size and
`maximizing yield and data utility.
`Prepared samples can be used on
`both single-read and paired-end
`fl ow cells.
`
`FULLY AUTOMATED SEQUENCING
`The multiplexed sequencing process
`is fully automated using the Genome
`Analyzer, Cluster Station, and Paired-
`End Module.
` The Cluster Station amplifi es DNA
`from up to 96 samples on the fl ow
`cell surface to create clusters
`containing 500–1,000 clonal copies of
`each molecule. The resulting
`high-density array of templates is
`sequenced using the Genome
`Analyzer and the Paired-End Module.
` Sequencing by synthesis is
`
`performed in parallel with novel
`fl uorescently labeled reversible ter-
`minator nucleotides. A total of three
`sequencing reads are performed
`(Figure 1). The fi rst read is identical to
`that of a paired-end experiment and
`uses the standard Read 1 Sequencing
`Primer provided in the Paired-End
`Cluster Generation Kit. At the end of
`the fi rst read, the extended sequenc-
`ing primer is removed and the Index
`Sequencing Primer, provided in the
`Multiplexing Sequencing Primers and
`PhiX Control Kit, is annealed to the
`same strand. This approach lever-
`ages the Paired-End Module to avoid
`the loss of high-quality sequencing
`data from the unknown sample that
`would occur if the index sequence
`had been included at the start of an
`application read.
`
`FIGURE 2: THE MULTIPLEXED SEQUENCING SAMPLE PREPARATION METHOD
`FOLLOWS THE FAMILIAR PAIRED-END PROTOCOL
`
`Purified DNA fragments (< 800 bp)
`
`Repair ends
`
`Blunt-ended fragments with
`5'-phosphorylated ends
`
`3’-dA overhang
`
`Adapter-modified ends
`
`Removal of unligated
`adapters
`
`Indexed DNA library
`
`Add an ‘A’ to the 3’ ends
`
`Ligate novel paired-end adapter
`
`Purify ligation product
`
`PCR using two novel universal
`primers plus index primer
`
`The multiplexed sequencing sample preparation method follows standard sample
`preparation protocols for paired-end sequencing, with the exception of using a
`novel adapter and PCR primers (shown in red).
`
`Ariosa Exhibit 1010, pg. 2
`IPR2013-00276
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`

`ILLUMINA® SEQUENCING
`
`FIGURE 3: ADDING THE SEQUENCE INDEX TO A LIBRARY
`
`Rd1 SP
`
`DNA Insert
`
`1. During sample preparation, adapters are ligated to the DNA fragments.
`One adapter contains the sequencing primer site for application read 1
`(Rd1 SP).
`
`P5
`
`Index SP
`
`Rd2 SP
`
`2. Prepared samples are amplified via PCR using two universal primers. One
`primer contains an attachment site (P5) for the flow cell, while the other
`contains the sequencing primer sites for the index read (Index SP) and
`for application read 2 (Rd2 SP).
`
`Index
`
`P7
`
`3. A third primer in the PCR adds the Index as well as a second flow cell
`attachment site (P7) to the PCR product shown in step 2.
`
`P5
`
`Rd1 SP
`
`DNA Insert
`
`Index SP
`
`Index
`
`P7
`
`4. The indexed library is ready for sequencing using the Genome Analyzer
`system.
`
`Rd2 SP
`
`detecting structural variation, and
`assembling sequences de novo.
` Like all of Illumina’s software
`solutions, Pipeline software offers
`an open architecture that allows for
`easy customization of downstream
`analysis and integration with a vari-
`ety of innovative analysis tools.
`
`UNLIMITED ACCESSIBILITY
`The Multiplexing Sample Preparation
`Oligonucleotide Kit and Multiplexing
`Sequencing Primers and PhiX Control
`Kit simplify high-throughput
`multiplexed sequencing with the
`Genome Analyzer system. In addition
`to multiplexing, the Genome
`
` Prior to application read 2, the
`index sequencing product is removed
`and the clusters are modifi ed in situ
`to regenerate the complementary
`strand using the Paired-End Module.
`The Multiplexing Read 2 Sequencing
`Primer is annealed to the comple-
`mentary strand and extended to
`complete the fi nal read.
` 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
`with industry-leading accuracy.
`Data quality is equivalent to that
`routinely achieved in a single-read
`or paired-end run. Due to the inher-
`ent redundancy in the index design,
`both perfect index reads and those
`that differ by one base can be used
`as sample identifi ers (Figure 4).
`
`AUTOMATIC SAMPLE IDENTIFICATION
`AND PROCESSING
`Pipeline Analysis software (version 1.0
`and higher) includes the ability to
`discriminate between the three
`reads. Once the sequencing chemistry
`is complete, the alignment software
`identifi es the index sequence and
`annotates each read with its
`respective index. From this point
`on, reads derived from a given
`multiplexed sample can be positively
`identifi ed.
`
`Just as in a non-multiplexed
`sequencing run, Pipeline software
`provides automated base-calling,
`calculation of quality values for
`every base, and alignment of read
`pairs to a reference sequence. The
`ability to combine multiplexing
`with paired-end reads is crucial
`for optimizing sequence alignment,
`
`Ariosa Exhibit 1010, pg. 3
`IPR2013-00276
`
`

`

`ILLUMINA® SEQUENCING
`
`Analyzer system can be used for
`single-read sequencing, mRNA-Seq,
`ChIP-Seq studies, and more. As an
`open platform for genetic analysis,
`the Genome Analyzer enables the
`broadest range of applications. View
`the latest accomplishments using
`Illumina sequencing technology at
`www.illumina.com/publications.
`
`FIGURE 4: PERCENTAGE OF USABLE INDEX READS
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`7
`Library Number
`
`8
`
`9
`
`10
`
`11
`
`12
`
`Perfect index reads
`
`Single-error index reads
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`Percent of Usable 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
`
`Multiplexing Sample Preparation
`Oligonucleotide Kit
`
`Kitted oligonucleotides used to prepare up to
`96 samples for multiplexed sequencing*.
`
`Multiplexing Sequencing Primers
`and PhiX Control Kit
`
`Kitted multiplexing sequencing primers, multiplexing
`control DNA, and buffer set. Suffi cient for up to
`10 Genome Analyzer runs.
`
`CATALOG NO.
`
`PE-400-1001
`
`PE-400-1002
`
`*Requires Genomic DNA or Paired-End Sample Prep Kits, available separately.
`
`ADDITIONAL INFORMATION
`Visit our website or contact us at the
`address at right to learn more about
`Illumina sequencing applications.
`
`Illumina, Inc.
`Customer Solutions
`9885 Towne Centre Drive
`San Diego, CA 92121-1975
`1.800.809.4566 (toll free)
`1.858.202.4566 (outside the U.S.)
`techsupport@illumina.com
`www.illumina.com
`
` FOR RESEARCH USE ONLY
`
`© 2008 Illumina, Inc. All rights reserved.
`Illumina, Solexa, Making Sense Out of Life, Oligator, Sentrix, GoldenGate, DASL, BeadArray, Array of Arrays, Infinium, BeadXpress, VeraCode, IntelliHyb, iSelect, CSPro,
`iScan, and GenomeStudio are registered trademarks or trademarks of Illumina. All other brands and names contained herein are the property of their respective owners.
`Pub. No. 770-2008-011 Current as of 2 December 2008
`
`Ariosa Exhibit 1010, pg. 4
`IPR2013-00276
`
`