Customers can design custom GoldenGate Assays to target specific SNP loci for their organism of study. Illumina scientists and a proprietary Assay Design Tool (ADT) help customers create successful content panels. The ADT provides a simple and powerful method for evaluating individual loci and creating the most successful custom genotyping assays.

Researchers create custom panels by selecting and submitting a requested list of loci to Illumina, which is evaluated with the ADT. Custom content designs can be submitted to a web application using sequence, accession number, gene symbol, gene ID, GI number, or chromosomal region. The SNPScore file provides predicted success information, validation status, and minor allele frequencies from published studies. Researchers use the ADT SNPScore file output to refine an initial assay panel to include desired assays that are predicted to have a high likelihood of success. This information can be used to reject some SNPs and ultimately create a final SNPScore file for the order.

• The GoldenGate Assay may be deployed on BeadArray or BeadXpress platforms
• The BeadArray platform supports the highest multiplex levels, while the BeadXpress platform is an ideal option for high-throughput biomarker screening

The GoldenGate Assay allows for a high degree of loci multiplexing during the extension and amplification steps, minimizing time, reagent volumes, and material requirements of the process.

• The DNA sample used in this assay is activated for binding to paramagnetic particles
• This activation step is a highly robust process that requires a minimum input of DNA (250ng at 50ng/μl. Depending upon the multiplex level, this equates to only 160pg of DNA per SNP genotype call
• Assay oligonucleotides, hybridization buffer, and paramagnetic particles are combined with the activated DNA in the hybridization step

The panel also contains Ancestry Informative Markers (AIMs) chosen from three populations that may be used to assess potential stratification in study samples.

• SNP sequences are optimized for Illumina's proven GoldenGate Genotyping Assay protocol

The mouse has been increasingly recognized as an optimal animal for the study of the genetics of complex disease traits. Two Mouse Linkage Panels (Mouse Low Density [LD] Linkage and Mouse Medium Density [MD] Linkage) are available for mouse genetics applications. These single nucleotide polymorphism (SNP) panels offer a low-cost, efficient method for attaining genome-wide genetic data to identify quantitative trait loci (QTLs), map candidate genes, and increase genotyping throughput.

The panel loci were chosen to maximize genetic information across the top ten inbred strains at a higher resolution than the Mouse LD Linkage Panel. SNPs in this panel were chosen to first provide uniform genome distribution at a density of approximately three SNPs per 5 Mb intervals across the genome. At least one SNP per interval was chosen to be informative for crosses involving the C57Bl/6J strain, and the remaining two SNPs were selected based on allele frequency and optimal spacing. On average, at least one SNP in each interval is informative for 75% of pairwise strain combinations.

• The Mouse LD Linkage Panel can be used on 12- and 32-sample Universal BeadChips
• SNP sequences are optimized for Illumina's proven GoldenGate Genotyping Assay protocol

The mouse has been increasingly recognized as an optimal animal for the study of the genetics of complex disease traits. Two Mouse Linkage Panels (Mouse Low Density [LD] Linkage and Mouse Medium Density [MD] Linkage) are available for mouse genetics applications. These single nucleotide polymorphism (SNP) panels offer a low-cost, efficient, method for attaining genome-wide genetic data to identify quantitative trait loci (QTL), map candidate genes, and increase genotyping throughput.

This panel has been optimized for application to N2 and F2 mouse genetics crosses, those typically used for mapping QTLs. It provides approximately 175-200 informative markers per cross and covers the entire mouse genome.

The panel was designed to include approximately four SNPs per 27 Mb interval. At least one SNP in each interval was chosen to be informative in crosses involving the C57Bl/6J strain. On average, at least one SNP in each interval is informative for 85% of all possible strain combinations.

• The Mouse LD Linkage Panel can be used on 12- and 32-sample Universal BeadChips
• SNP sequences are optimized for Illumina's proven GoldenGate Genotyping Assay protocol

The cDNA Synthesis Kit is used in the first step of the DASL Assay protocol where total RNA is converted to cDNA using both biotinylated nonamers (biotin-d(N)9) and biotinylated oligo d(T)18, cleaned up and made ready for use in the GoldenGate Assay amplification step.

The HumanMethylation27 panel targets CpG sites located within the proximal promoter regions of transcription start sites of 14,475 consensus coding sequencing (CCDS) in the NCBI Database (Genome Build 36). In addition, 254 assays cover 110 miRNA promoters. On average, two assays were selected per CCDS gene and from 3-20 CpG sites for >200 cancer-related and imprinted genes.

Unlike other genome-wide methylation arrays, the Infinium HumanMethylation27 BeadChip enables single CpG-site resolution, higher sample throughput (up to 12 samples per array), and requires as little as 500 ng of bisulfite-converted DNA per sample. The Infinium DNA methylation platform is highly suitable for detailing the biological role of DNA methylation in both normal and diseased cells, and for novel DNA methylation marker discovery.

• Methylation profiles can be compared between different samples and results can easily be integrated with RNA expression profiling experiments using GenomeStudio software

The content is derived from the National Center for Biotechnology Information Reference Sequence (NCBI RefSeq) database (Build 36, Release 22), supplemented with probes derived from the Mouse Exonic Evidence Based Oligonucliotide (MEEBO) set, as well as exemplar protein-coding sequences described in the RIKEN FANTOM2 database. This BeadChip targets approximately 25,600 well-annotated RefSeq transcripts, over 19,100 unique genes, and enables the interrogation of eight samples in parallel.

The BeadChip content is derived from the National Center for Biotechnology Information Reference Sequence (NCBI RefSeq) database (Build 36, Release 22), supplemented with probes derived from the Mouse Exonic Evidence Based Oligonucliotide (MEEBO) set as well as exemplar protein-coding sequences described in the RIKEN FANTOM2 database.

• The MouseWG-6 BeadChip contains the full set of MouseRef-8 BeadChip probes with an additional 11,603 probes from the above databases

The kit includes sufficient hybridization buffer, wash buffer, and trays to process six BeadChips.

The probe content sources include the National Center for Biotechnology Information (NCBI) Reference Sequence Database (Releases 16 NM, 16 XM, and 16XR), and UniGene.

The assay combines the unique PCR and labeling steps from Illumina's DASL Assay with gene-based hybridization and the whole-genome probe set of its Direct Hybridization Assay (Human HT-12 v4 Expression BeadChip). This greatly increases the DASL assay target set, which consists of well-characterized NCBI RefSeq genes (RefSeq Build 36.2, Release 38), while retaining the ability to accurately profile partially-degraded RNA samples. Highly reproducible expression profiles (r² >0.97) can be produced from as little as 10–100 ng total RNA from fresh frozen tissue or 50–200 ng total RNA from FFPE samples.

The TruSeq SR Cluster Kit v3-cBot-HS contains cluster generation reagents for the cBot cluster amplification system in a pre-mixed, 96-well plate format that requires minimal reagent preparation. DNA library samples are bound to complementary adapter oligos grafted on the surface of the flow cell. The templates are copied from the hybridized primer by 3' extension using a high fidelity DNA polymerase. These copies are isothermally amplified to create clonal clusters of ~1,000 copies each, ready for sequencing.

The streamlined, automated protocol allows researchers to screen up to 16 times as many samples per reaction as the standard GoldenGate Assay, increasing throughput from 288 samples per day to greater than 2000, while decreasing total reagent consumption. Current plexity ranges for GoldenGate Indexing include 96-plex, 192-plex, and 384-plex. The GoldenGate Indexing Assay is conveniently processed on the Universal-32 BeadChip.

• GoldenGate Indexing is ideal for agricultural researchers, as well as researchers from other areas of interest, that have a need for efficient, low-plex, high-sample throughput genotype screening. Illumina's agriculture solutions.

The TruSeq Exome Enrichment Kit supports cost-effective, scalable exome sequencing studies, offering pre-enrichment sample pooling and the most comprehensive exome coverage, highest uniformity, and lowest DNA input requirement. Master-mixed reagents are coupled with plate-based processing for up to 96 samples and volumes are optimized for liquid handling robots, making the process automation-friendly for even higher throughput.

Prior to exome enrichment, libraries are prepared using the TruSeq DNA Sample Prep Kit, which provides a robust indexing solution for each DNA sample, enabling multi-sample pooling of up to six samples in a single enrichment reaction. Multi-sample pooling dramatically reduces hands-on time compared to other available methods, making large high-throughput studies feasible and economical.

*Limited-time offer
TruSeq Exome Enrichment—starting at just $49 USD per sample! Contact your sales representative to order today.

*Pricing valid for US customers only.  Promotional pricing for customers outside the US and in distributor countries will vary.

Featured Researchers

“HiSeq has been a game changer for us. A project that would have taken us a year, now takes one week.”

—GRAHAM WILEY, Ph.D.
Associate Research Scientist, Arthritis & Clinical Immunology Research Program,
Oklahoma Medical Research Foundation

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“With the HiSeq, we can sequence deeply enough that we’re not only able to discover mutations, but we can also find very rare mutations.”

—SETH CROSBY, M.D.
Director, Genome Technology Access Center,
Washington University at St. Louis

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Workflow improvements enable streamlined sample preparation, allowing economical studies covering all small RNA transcripts in any species. Illumina's unique combination of long and short reads, single and paired-end sequencing, and capacity for tens of millions to billions of reads per run enables researchers to:

• Annotate coding SNPs
• Discover transcript isoforms
• Identify regulatory RNAs
• Characterize splice junctions
• Determine the relative abundance of transcripts

Learn more about TruSeq technology.

Custom oligonucleotide probes are first designed and ordered in DesignStudio. The TruSeq Custom Oligo Set consists of 2,500 to 67,200 attempted custom oligos. TruSeq Custom Enrichment is fully integrated with TruSeq DNA Sample Preparation Kits for an optimized, gel-free workflow.

Based on the total number of samples requested, the TruSeq Custom Oligo Set  includes sufficient 4-, 8- , 24-, or 96-reaction TruSeq Custom Enrichment reagent kits. Through pre-enrichment sample pooling of up to 12 samples, the 4-reaction kit supports 48 samples, the 8-reaction kit supports 96 samples, the 24-reaction kit supports  288 samples, and the  96-reaction kit supports  1,152 samples.

*Limited-time offer

Get a 25% discount on the TruSeq Custom Enrichment 8-reaction, 96 Sample Kit. Contact your sales representative to order today.
*Promotional pricing for customers outside the US, Europe, and in distributor countries (including European countries covered by a distributor) may vary. No quantity restrictions. Not valid with other discounts.

Start a new TruSeq Custom Enrichment project »
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Learn more about TruSeq technology.

Chromatin immunoprecipitation (ChIP) is a powerful method to selectively enrich for DNA sequences bound by a particular protein in living cells. ChIP-Seq on Illumina sequencing systems supports virtually unconstrained selection of any ChIP-able protein and/or modification to be studied. These include transcription factors, polymerases and transcriptional machinery, structural proteins, protein modifications, and DNA modifications. The ChIP-Seq DNA Sample Prep Kit is used to build DNA libraries for single-read sequencing.

The ChIP process enriches specific crosslinked DNA protein complexes using an antibody against a protein of interest. Unique oligonucleotide adapters are then added to the small stretches of DNA that were bound to the protein of interest to enable massively parallel sequencing. Following a short PCR enrichment, the library is ready to load on cBot or Cluster Station for cluster generation.

Following DNA fragmentation, 2-5 Kb fragments are end-repaired with biotin labeled dNTPs. The DNA fragments are circularized, and non-circularized DNA is removed by digestion. Circular DNA is fragmented and biotin labels of the fragments (corresponding to the ends of the original DNA ligated together) are affinity purified. Purified fragments are end-repaired and ligated to Illumina Paired-End sequencing adapters.

Additional sequences complementary to the flow cell oligonucleotides are added to the adapter sequence with tailed PCR primers. The final prepared libraries consist of short fragments made up of two DNA segments that were originally separated by several kilobases. These libraries are ready to load on cBot or the Cluster Station for cluster generation.

The industry's fastest sample prep.

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Workflow shown using the Nextera Sample Preparation Kit, and 1 x 36 bp sequencing on MiSeq system.

With Nextera technology, DNA is simultaneously fragmented and tagged with sequencing adapters in a single step, using standard lab equipment. Ideal for precious samples available in limited quantity, the protocol requires only 50 ng of DNA input. Libraries prepared with Nextera kits are compatible with all Illumina sequencers.

The TruSeq Dual Index Sequencing Primer Box is required for single-read and paired-end sequencing of Nextera libraries on HiSeq 1000/2000, HiScanSQ, and Genome Analyzer IIx instruments. These primers are required for non-indexed, single-indexed, and dual-indexed Nextera libraries.

The kit selectively targets microRNA, or other polycistronic RNAs that have been processed by DICER or similar nucleases, which provide a distinct molecular signature. The RNA fragments are ligated to Illumina adapters followed by reverse transcription and PCR amplification. These libraries are ready to load on cBot or the Cluster Station for cluster generation.

Master-mixed reagents eliminate the majority of pipetting steps and reduce the amount of clean-up, as compared to previous methods, minimizing hands-on time. This results in economical, high-throughput RNA sequencing studies achieved with the easiest-to-use sample preparation workflow offered by any NGS platform.

Learn more about TruSeq technology.

Master-mixed reagents eliminate the majority of pipetting steps and reduce the amount of clean-up, as compared to previous methods, minimizing hands-on time. This results in economical, high-throughput DNA sequencing studies achieved with the easiest-to-use sample preparation workflow offered by any sequencing platform.

• The TruSeq DNA v2 Sample Preparation kit includes a gel-free protocol optimized for use with the TruSeq Exome Enrichment Kit and the TruSeq Custom Enrichment Kit

Learn more about TruSeq technology.

Available for single- or paired-end read sequencing, each box includes sufficient reagents for processing a single flow cell.

Ends are repaired with a combination of fill-in reactions and exonuclease activity to produce blunt ends. An 'A'- base is added to the blunt ends followed by ligation to Illumina Paired-End Sequencing adapters. These adapters contain two unique sequencing primer hybridization sites.

Additional sequences complementary to the oligonucleotides in the flow cell are added to the adapter sequences with tailed PCR primers, followed by gel-based size selection and purification. These libraries are ready to load on cBot or the Cluster Station for cluster generation.

This BeadChip was developed by Illumina in collaboration with the USDA-ARS, University of Missouri, and the University of Alberta. More than 24,000 SNP probes target novel SNP loci that were discovered by sequencing three pooled populations of economically important beef and dairy cattle using Illumina's Genome Analyzer. Additional content is derived from publicly available sources such as the bovine reference genome, Btau1, and the Bovine HapMap Consortium data set. All SNP probes have been validated in 19 common beef and dairy breeds. This product targets evenly distributed SNPs that are polymorphic across the breeds tested and provides an average probe spacing of 49.4kb and a median spacing of 36.9kb.

The BovineSNP50 BeadChip is a multi-sample genotyping panel powered by Illumina's Infinium HD Assay. This assay provides the industry's highest call rates, allows for flexible content deployment, and enables the detection and measurement of copy number variation.

• This 24-sample BeadChip represents the highest density genotyping solution for characterizing the genome in dairy and beef cattle
• The PCR-free, single tube sample preparation significantly reduces labor and potential sample handling errors
• Laboratory Information Management System (LIMS) and robotic automation are available to accurately and efficiently track samples throughout analysis

Supporting the most comprehensive genome-wide genotyping studies, the 777,000 SNP BovineHD BeadChip expands the diversity of bovine breeds assessed in genetic prediction and enables more discoveries of quantitative traits.

Featuring 6,909 SNPs uniformly distributed across the bovine genome, it provides a robust solution for high accuracy imputation to the BovineSNP50 BeadChip. As with all Illumina Infinium genotyping arrays, the BovineLD BeadChip can be customized with Add-On Content of 1,000 to 80,000 custom markers for ultimate design flexibility.

Illumina scientists and collaborators referenced historical data generated with the BovineSNP50 BeadChip to identify the best SNP content for imputation efficiency among global dairy breeds. In silico testing determined that the highest imputation efficiency could be achieved by optimizing minor allele frequencies (MAFs) among targeted breeds, evenly spacing SNPs across the entire bovine genome with higher marker densities at the chromosomal ends. Content captures all chromosomes, including X and mitochondrial DNA, and all known haplotypes of the Y chromosome. Uniform genomic coverage provides an average gap size of 383 kb and a median gap size of 347 kb. To ensure backward compatibility with the GoldenGate3K Bovine Array, Illumina retained 2,162 overlapping markers from the BovineLD BeadChip.

This multisample genotyping panel is powered by the widely used Infinium Assay, delivering the industry's highest call rates and reproducibility, along with precise detection. The assay's PCR-free single-tube sample preparation significantly reduces labor and potential sample handling errors. A multisample format further reduces experimental variability and overall project cost by allowing breeders to interrogate up to 24 samples in parallel.

Supporting the most comprehensive genome-wide genotyping studies, the 777,962 SNP BovineHD BeadChip expands the diversity of bovine breeds assessed in genetic prediction and enables more discoveries of quantitative traits.

Powered by Illumina's revolutionary Infinium Methylation Assay, this BeadChip allows researchers to interrogate > 485,000 methylation sites per sample at single-nucleotide resolution. Content was selected with the guidance of a consortium of methylation experts comprising 22 members that represent 19 institutions worldwide. It covers 99% of RefSeq genes, with an average of 17 CpG sites per gene region distributed across the promoter, 5'UTR, first exon, gene body, and 3'UTR. It covers 96% of CpG islands, with additional coverage in island shores and the regions flanking them. Further content categories requested by the Consortium include:

• CpG sites outside of CpG islands
• Non-CpG methylated sites identified in human stem cells
• Differentially methylated sites identified in tumor versus normal (multiple forms of cancer) and across several tissue types
• CpG islands outside of coding regions
• miRNA promoter regions

This BeadChip includes > 90% of the content contained on the HumanMethylation27 BeadChip. Its 12-sample per array format is compatible with automation, enabling up to 96 samples to be run in parallel (with full LIMS support available) and requiring only 500 ng of input (1 µg with automation). A protocol enabling compatibility with FFPE samples is now also available. This unique combination of features makes the HumanMethylation450 BeadChip a powerful tool to drive exciting new study designs and further fuel the rapid evolution of epigenetics research.

• Genotyping Studies with Illumina Omni BeadChips

The BeadChip was developed in collaboration with leading ovine researchers from AgResearch, Baylor UCSC, CSIRO, and the USDA as part of the International Sheep Genomics Consortium. It features over 54,241 evenly spaced probes that target single nucleotide polymorphisms (SNPs). More than 18,000 of these markers were discovered through sequencing reduced representation libraries with the Illumina Genome Analyzer IIx. A set of 600 SNPs were identified by BAC end sequencing and validated with Illumina GoldenGate Genotyping Assays over 403 animals from 23 breeds. The remaining SNPs were derived from the draft ovine genome.

The OvineSNP50 BeadChip provides uniform genome-wide coverage with an estimated one marker per 46kb on average. The BeadChip is powered by the Infinium HD Assay that provides the industry's highest call rates, allows for flexible content deployment, and enables the detection and measurement of copy number variation.

• The BeadChip further reduces experimental variability by allowing researchers to interrogate up to 12 samples in parallel
• The assay's single-tube sample preparation without PCR or ligation steps significantly reduces labor and potential sample handling errors

This BeadChip features 62,163 evenly spaced probes offering comprehensive coverage of the porcine genome. It was developed through Illumina's iSelect program in collaboration with leading porcine researchers from both US and International Universities and Research Institutes including Iowa State University, the University of Illinois, Cambridge University, and Wageningen University.

The Porcine SNP Chip Consortium made the final selection of the highest quality markers from more than 510,000 single nucleotide polymorphisms (SNPs), after combining novel discovered SNPs with several other existing databases and study results.

The panel was optimized using multiple criteria for marker selection, including minor allele frequency determined from representative sample sequencing, allele count, quality score, spacing, location, and validation status. More than 43,000 SNPs on the final panel were discovered using deep sequencing with the Illumina Genome Analyzer IIx.

Each array on the HumanHT-12 v4 Expression BeadChip targets more than 47,000 probes derived from the National Center for Biotechnology Information Reference Sequence (NCBI) RefSeq Release 38 (November 7, 2009) and other sources.

• 12-sample format enables large gene expression studies to be completed quickly and economically
• HumanHT-12 Expression BeadChips use the Direct Hybridization Assay and are scanned on the BeadArray Reader, iScan system, or HiScanSQ system.
• Sample labeling can be performed using the TargetAmp-Nano Labeling Kit for Illumina Expression BeadChip, available from Epicentre (an Illumina Company) or using the TotalPrep RNA Labeling Kit available from Ambion.

Featuring more than 777,000 SNPs that uniformly span the entire bovine genome, this BeadChip enables a broad range of applications such as genome-wide selection, identification of quantitative trait loci, evaluation of genetic merit, cross-breed mapping, linkage disequilibrium studies, comparative genetic studies, and breed characterization for evaluating biodiversity. The eight-sample BovineLD BeadChip, along with the proven Infinium HD Assay, presents a powerful high-throughput solution for whole-genome studies in cattle.

For high-throughput, cost-effective genetic screening, the BovineSNP50 BeadChip features more than 54,000 evenly spaced SNP probes spanning the bovine genome.

The highly polymorphic SNP content on the MaizeSNP50 BeadChip was subjected to rigorous functional testing across over 30 diverse maize lines to ensure strong performance. Importantly, this BeadChip presents an average of greater than 25 markers per megabase (Mb), providing ample SNP density for robust whole-genome genotyping studies. In addition, the MaizeSNP50 marker set increases the ability to perform genetic mapping and marker assisted breeding.

The MaizeSNP50 BeadChip is powered by the proven Infinium HD Assay, that provides the industry's highest call rates and allows for flexible content deployment.

• The assay's single-tube sample preparation without PCR or ligation steps significantly reduces labor and potential sample handling errors
• The BeadChip further reduces experimental variability by allowing researchers to interrogate up to 24 samples in parallel

This BeadChip contains more than 170,000 markers placed on the CanFam2.0 reference sequence. Illumina developed the BeadChip in collaboration with the LUPA Consortium, which includes 22 European universities and other partners such as the Broad Institute.

This Panel consists of 1,421 thoroughly screened and validated SNP loci from > 400 genes thought to be involved in cancer. Panel content was selected from the National Cancer Institute's Cancer Genome Anatomy Project SNP500 Cancer Database.

SNPs in the panel were chosen to be within 10kb of each gene and to represent several pathways thought to be involved in the etiology of various types of cancers including apoptosis, oncogenesis, tumor suppression, and G-protein coupled receptor protein signaling. The distribution of SNPs by gene region includes > 300 coding SNPs of which 158 cause amino acid changes in the resulting protein. In the Cancer SNP Panel, > 3 SNP assays were selected, on average, for each gene represented.

The SNP assays included on the Cancer SNP Panel were subjected to rigorous functional testing to ensure strong performance and suitability for association studies using Illumina's GoldenGate Assay. To determine call rate, reproducibility, and Mendelian inconsistencies, three populations were studied: Caucasians, Han Chinese and Japanese, and Yoruba Africans. Genotype concordance was assessed by comparing loci in the Illumina Cancer SNP Panel to genotype data from the SNP500 Cancer Database.

• SNP sequences are optimized for Illumina's proven GoldenGate Genotyping Assay protocol

• The Cancer SNP Panel can be deployed on 12-sample and 32-sample Universal BeadChips.

View the Omni Family of Microarrays »

View the Omni Family of Microarrays »

View the Omni Family of Microarrays »

View the Omni Family of Microarrays »

This BeadChip includes a complete panel of genome-wide tag SNPs and markers targeting all regions of known cytogenetic importance. It incorporates 200,000 “best of the best” SNPs with the highest tagging power.

220,000 markers provide useful content for cytogenetic analysis. This includes dense coverage of around 250 genomic regions commonly screened in cytogenetics laboratories, including subtelomeric regions, pericentromeric regions, sex chromosomes, and targeted coverage in around 400 additional disease-related genes.

Downstream data analysis is fully supported with Illumina's KaryoStudio and GenomeStudio software.

• Process up to 12 samples in parallel, thus increasing sample throughput and decreasing experimental variability
• Process samples using the single-tube, PCR-free Infinium HD Assay that requires only 200ng DNA per sample.

The TruSeq PE Cluster Kit v3-cBot-HS provides reagents for the cBot cluster amplification system. DNA library samples are bound to complementary adapter oligos grafted on the surface of the flow cell. The templates are copied from the hybridized primer by 3' extension using a high fidelity DNA polymerase. These copies are isothermally amplified to create clonal clusters of ~1,000 copies each, ready for sequencing.

Cluster generation reagents are provided in a pre-mixed, 96-well plate format that requires minimal reagent preparation. The kit also includes reagents for cluster resynthesis of the reverse strand, regenerated by bridge amplification within the paired-end flow cell. After resynthesis of the reverse strand, the original forward strand is cleaved and the reverse strand is sequenced for the second read.