Nextera DNA Flex Library Prep on the iSeq 100 System

This resource provides step-by-step guidance through the process of transitioning to Nextera DNA Flex Library Prep on the iSeq 100 System.

Initial Considerations

Total Library Prep Time

The total library prep time for Nextera DNA Flex is 2.5 hours.

For a comparison of the Nextera DNA Flex Library Prep time to TruSeq DNA Nano Library Prep (6 hours) and Nextera XT Library Prep (4 hours), see the Nextera DNA Flex Library Preparation Kit data sheet.

Comparing Nextera DNA Flex and Other Illumina DNA Library Prep Kits

The following data sheets provide information on the Nextera DNA Flex Library Prep kit compared with other Illumina DNA library prep kits.

Sequencing Methods

Refer to the following resources for guidance on sequencing methods using Nextera DNA Flex Library Prep and the iSeq 100 Sequencing System, including viral and bacterial whole-genome sequencing.

Assay Cost per Sample

Contact your local sales representative for help calculating the cost per sample.

Preparing Samples

Compatible Sample Types

Nextera DNA Flex supports the following sample types:

  • Small genomes
  • Large genomes
  • Blood
  • Saliva
  • PCR amplicons > 150 bp

­Nextera DNA Flex is optimized for gDNA (dsDNA) and is not compatible with ssDNA/RNA as these are not substrates for tagmentation. For more information on the supported sample types and requirements, see the DNA Input Recommendations section of the Nextera DNA Flex Library Prep Reference Guide, the Nextera DNA Flex Microbial Colony Extraction Demonstrated Protocol Guide (colonies), or Nextera Crude Lysate Protocol for Metagenomic Next-Generation Sequencing (stool samples).

Using PCR Amplicons as Input

PCR amplicons > 150 bp can be used as input. For additional information, refer to the Sample Input Recommendations section of the Nextera DNA Flex Library Prep Reference Guide.

Input DNA Requirements

The Nextera DNA Flex protocol is compatible with DNA inputs 1–500 ng. For human DNA samples and other large complex genomes, the recommended DNA input is 100–500 ng. For small genomes, the DNA input amount can be reduced to as low as 1 ng (modifying the PCR cycling conditions accordingly). Note that if using < 100 ng DNA input, quantifying and normalizing the initial DNA sample is required.

For more information on DNA input recommendations, see the Nextera DNA Flex Library Prep Reference Guide and the Nextera DNA Flex FAQs.

For specific guidance regarding microbial colonies, see the Microbial Whole-Genome Sequencing with Nextera DNA Flex Library Preparation Kit application note.

Quantifying Samples

For DNA inputs between 100–500 ng, accurate quantification of the initial DNA sample is not required, and normalization of the final yield is expected.

If you are using < 100 ng DNA input, quantifying the initial DNA sample to determine the number of PCR cycles required is recommended. Use a fluorometric-based method to quantify double-stranded DNA, such as QuantiFluor or PicoGreen.

The concentration of gDNA can be determined using the Qubit dsDNA BR Assay or the Qubit dsDNA HS assay. These assays use a fluorescent dye that is highly selective for double-stranded DNA over RNA and can detect samples in a concentration range from 10 pg/μl – 1000 ng/μl. PicoGreen dye can also be used to accurately measure the DNA concentration.

For more information, see the DNA Input Recommendations section of the Nextera DNA Flex Library Prep Reference Guide.

Hybridizing Direct Microbial Colonies

The following have been tested using the Nextera DNA Flex Microbial Colony Extraction Demonstrated Protocol Guide:

  • Pseudomonas aeruginosa
  • Klebsiella pneumoniae
  • Enterobacter cloacae
  • Escherichia coli, Acinetobacter baumannii
  • Enterococcus faecalis
  • Streptococcus agalactiae
  • Staphylococcus aureus
Assessing DNA Quality

For information on assessing DNA sample quality, see the Assess DNA Purity section of the Nextera DNA Flex Reference Guide.

Using GC-rich or AT-rich Genomes

A variety of genomes prepared with Nextera DNA Flex (from bacteria, plants, agriculture, and human) can be found in BaseSpace Sequence Hub. The data remains consistent across genomes. Public data are available for the following systems:

  • MiSeq Sequencing System: Nextera DNA Flex (replicates of E. coli, B. cereus, and R. sphaeroides)
  • MiniSeq Sequencing System: Nextera DNA Flex (replicates of E. coli, B. cereus, and R. sphaeroides)
Assessing DNA Purity

The ratio of absorbance at 260 nm to absorbance at 280 nm provides an indication of sample purity. This protocol is optimized for DNA with absorbance ratio values of 1.8–2.0, which indicates a pure DNA sample. Values outside this range indicate the presence of contaminants that may cause incomplete tagmentation and adversely impact the final library yield. For a complete list of contaminants, including sources, avoidance, and effects on the library, see the Nextera XT Troubleshooting Technical Note.

Incomplete tagmentation caused by contaminants might result in library prep failure, poor clustering, or an unexpectedly high scaffold number.

For more information, see the Assess DNA Purity section of the Nextera DNA Flex Reference Guide.

Preparing Libraries Using Nextera DNA Flex

Overview of Nextera DNA Flex

For an introduction to using the Nextera DNA Flex Library Prep kit, view the following resources:

Required Reagents and Equipment

For information on the required kits, consumables, and equipment, see the Nextera DNA Flex Consumables & Equipment List. Nextera DNA Flex reagents are not compatible with Nextera DNA or Nextera XT reagents.

Index adapters and library prep kits are sold separately. If starting the protocol from blood samples, the Flex Lysis Reagent kit is also required. Using third-party indexes is not recommended.

Differences in Kit Components Between Nextera DNA Flex, Nextera XT, and TruSeq DNA Nano Library Prep Kits

Between the Nextera XT and Nextera DNA Flex kits there is one reagent in common: Resuspension Buffer.

Between the Nextera DNA Flex and TruSeq DNA Nano kits, there are three reagents in common: Resuspension Buffer, Enhanced PCR Mix, and the Sample Purification Beads.

For more information, see Nextera DNA Flex Kit Contents, Nextera XT Kit Contents, and TruSeq DNA Nano Kit Contents.

Components to Order Separately

Refer to the Nextera DNA Flex Library Prep Consumables and Equipment List. This information is also available in the Kit Contents section of the Nextera DNA Flex Library Prep Reference Guide.

Checking Library Quality

Use a Fragment Analyzer (Agilent, formerly Advanced Analytical) or Agilent Technologies 2100 Bioanalyzer to check the quality and intended size distribution of a tagmented sample. For examples of Bioanalyzer traces and library size distributions, see the Nextera DNA Flex Library Prep Reference Guide. Variation in the Bioanalyzer profiles is expected because it is dependent on the input DNA type.

For more information, see the Check Library Quality section of the Nextera DNA Flex Library Prep Reference Guide. The following video resources are also available:

Direct Bacterial Colony Sequencing and Microbial Whole-Genome Sequencing

The following resources are available:

Differences Between Nextera DNA Flex and Nextera Library Prep Kits

See above under Initial Considerations.

Comparison of Nextera DNA Flex and TruSeq DNA Nano Sequencing Metrics

See the Nextera DNA Library Prep Performance Table in the Nextera DNA Flex Library Prep Kit Data Sheet.

Recommended Dilution Procedure

For Nextera DNA Flex, the loading volume is 20 μl and the loading concentration is 200 pM. Library is automatically denatured into single strands and further diluted onboard the instrument.

For more information, see the Prepare the Flow Cell and Libraries and Cluster Generation sections in the iSeq 100 Sequencing System Guide.

Index Adapters Used With Nextera DNA Flex

This kit uses optimized Nextera adapters.

For more information, see the Sequences for Nextera Kits section of the Illumina Adapter Sequences Document and the Pooling Guidelines for Nextera Kits section of the Index Adapters Pooling Guide

Normalization

The Nextera DNA Flex Library Prep kit uses a bead-based transposome complex to tagment genomic DNA by fragmenting and adding adapter tag sequences in a single reaction step. When saturated with input DNA, the bead-based transposome complex fragments a set number of DNA molecules, providing flexibility to use a wide DNA input range, consistent tight fragment size distribution, and normalized libraries. Following the tagmentation step, a limited-cycle PCR step adds Nextera DNA Flex-specific index adapter sequences to the ends of a DNA fragment, enabling capability across all Illumina sequencing platforms. A subsequent Sample Purification Bead (SPB) cleanup step then purifies libraries for use on an Illumina sequencing system.

For more information, see the How the Nextera DNA Flex Assay Works section in the Nextera DNA Flex Library Prep Reference Guide.

Direct Bacterial Colony Sequencing

Refer to the Demonstrated Protocol for Nextera DNA Flex Microbial Colony Extraction and the Direct Bacterial Colony Sequencing with the Nextera DNA Flex Library Prep Kit application note.

The following literature references from the application note may also be helpful:

  • Reuter S, Ellington MJ, Cartwright EJ, et al. Rapid bacterial whole-genome sequencing to enhance diagnostic and public health microbiology. JAMA Intern Med. 2013;173:1397–404.
  • Quainoo S, Coolen JPM, van Hijum SAFT, et al. Whole-genome sequencing of bacterial pathogens: the future of nosocomial outbreak analysis. Clin Microbiol Rev. 2017;30:1015–1063.
  • Rubin BE, Sanders JG, Hampton-Marcell J, et al. DNA extraction protocols cause differences in 16S rRNA amplicon sequencing efficiency but not in community profile composition or structure. MicrobiologyOpen. 2014;3:910–921.
  • van Tongeren SP, Degener JE, Harmsen HJM. Comparison of three rapid and easy bacterial DNA extraction methods for use with quantitative real-time PCR. Eur J Clin Microbiol Infect Dis. 2011;30:1053–1061.
  • Vesty A, Biswas K, Taylor MW, Gear K, DouglasRG. Evaluating the impact of DNA extraction method on the representation of human oral bacterial and fungal communities. PLoS One. 2017;12:e0169877.
  • Illumina (2017). Nextera DNA Flex Library Prep Kit data sheet. Accessed January 5, 2018.
  • NextSeq Control Software and Real-Time Analysis v2 Software. support.illumina.com/sequencing/sequencing_instruments/nextseq500.html. Accessed January 9, 2018.
  • Carver T, Harris SR, Berriman M, Parkhill J and McQuillan JA. Artemis: an integrated platform for visualization and analysis of high-throughput sequence-based experimental data. Bioinformatics 2012;28;464–469.
  • Bankevich A, Nurk S, Antipov D, et al. SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol. 2012; 19: 455–477.
  • Gurevich A, Saveliev V, Vyahhi N, Tesler G. QUAST: quality assessment tool for genome assemblies. Bioinformatics. 2013;29:1072–5.
  • Miller JR, Koren S, Sutton G. Assembly algorithms for next-generation sequencing data. Genomics. 2010;95:315–327
Factors Inhibiting Library Prep

Several factors can impair the performance of the Nextera enzyme:

  • Proteins can coat DNA, preventing enzyme binding to the substrate.
  • Sequestration of enzyme cofactors by EDTA can negatively affect enzyme function.
  • Proteinases, detergents, and phenol can degrade the enzyme.
  • Changes in ionic strength and pH caused by chemicals left over from library prep.
  • Some sample types might be more difficult to work with (eg, fecal, environment) due to additional inhibitors that are not entirely removed during DNA extraction. Consider using additional cleanup, such as bead or column cleanup.

For additional information, see Nextera XT Library Prep: Tips and Troubleshooting.

Extracting Gram Positive Bacteria DNA

For information on extraction protocol, see the Nextera DNA Flex Microbial Colony Extraction Protocol Guide and the Direct Bacterial Colony Sequencing with the Nextera DNA Flex Library Prep Kit application note. The application note includes additional resources.

Sample Indexing

Indexing is a way to label biological samples, then use bioinformatic methods to distinguish the sequencing data generated by each during a run. 

For more information, see the Dual-Indexed Workflow on a Paired-End Flow Cell (Workflow B) section of the Indexed Sequencing Overview Guide.

Multiplexing Samples

1–96 samples can be processed at a time through the protocol. However, it is recommended to process 8 samples or multiples of 8 using a multichannel pipette.

To optimize sample pooling for your experiment, you need to know the sample genome size, your desired coverage, and the duplicate percentage. Further information regarding multiplexing considerations can be found under Optimizing Sample Pooling in the Sequencing Nextera DNA Flex on the iSeq 100 System sections below.

Selecting Index Combinations

For information on the supported index combinations, including in cases for which only a small number of samples will be pooled, see the Nextera Pooling Guidelines section of the Index Adapters Pooling Guide or the Pooling Calculator.

Use of Own Indexes During PCR

It is not recommended to use any other indexes as the indexes in the kit have high purity and have been carefully optimized to result in even index representation across libraries.

For more information, see the Illumina Nextera Adapters section of the Illumina Adapter Sequences document and the Nextera Pooling Guidelines section of the Index Adapters Pooling Guide.

Differences Between SPB and SPRI

SPB is the acronym for Sample Purification Beads and SPRI is the acronym for Solid Phase Reversible Immobilization. These two terms are synonymous. Illumina has transitioned from using the SPRI terminology to SPB.

Double-sided Bead Purification

The purpose of the double-sided bead purification is to size select the library fragments. The two-step process first removes the large fragments and the second step removes the small molecular weight fragments.

For more detailed information on sample purification bead size selection and best practices, see the online training sequencing course TruSeq: Sample Purification Bead Size Selection and Best Practices.

It is not recommended to use third-party sample purification beads for cleaning up libraries. Third-party beads or columns might not be compatible with the Nextera DNA Flex Library Prep kit.

Compatible Thermal Cyclers

Coverage of GC regions can be impacted by the model, settings, and performance of the thermal cycler used. Illumina has validated the Bio-Rad DNA Engine Tetrad 2, the Bio-Rad S1000, the Bio-Rad C1000, and the MJ Research PTC-225 DNA Engine Tetrad thermal cyclers. Other thermal cyclers may differ in performance, which can impact genomic coverage.

Also see the Nextera DNA Flex Consumables & Equipment List.

Compatible Indexes and Sequencing Primers

Nextera DNA Flex is compatible with the Nextera CD indexes and the IDT for Illumina Nextera UD Index kits. It is not compatible with Nextera XT v2 Index kits.

On-Bead Tagmentation

For information on on-bead tagmentation, refer to the following resources: 

Additional Guidance on Best Practices for the Nextera Flex Library Prep Kit

The following resources are available:

Sequencing Nextera DNA Flex on the iSeq 100 System

Sequencing by Synthesis (SBS) Technology

For information on SBS technology, view Sequencing: Illumina Technology video or refer to An Introduction to Next-Generation Sequencing Technology.

Required Reagents and Equipment

The iSeq 100 System requires the iSeq 100 i1 Reagents kit. For information on required user-supplied consumables and equipment, see the iSeq 100 Sequencing System Guide.

Information on the iSeq 100 System

Documentation and Training Videos can be found on the iSeq 100 Sequencing System support page.

Setting up a Sequencing Run

Refer to the Set Up a Sequencing Run (Local Run Manager Mode) or Set Up a Sequencing Run (Manual Mode) sections of the iSeq 100 Sequencing System Guide.

Optimizing Sample Pooling

To optimize sample pooling for your experiment, you need to know the genome size of your sample, the coverage you desire, and the duplicate percentage. The recommended read length for Nextera DNA Flex libraries is 151 cycles. The Sequencing Coverage Calculator can calculate the number of samples that can be pooled.

Refer to the Estimating Sequencing Coverage Technical Note for more guidance on planning your sequencing run. 

The following resources are also available:

Determining the Recommended Sequencing Depth

The optimal sequencing depth varies depending on the application you are running and your experimental goals. Refer to the literature for applicable reference studies.

Determining the Required Read Length

If you are using Nextera DNA CD indexes, 2x151 bp is recommended. If you are using IDT for Illumina Nextera UD Indexes, refer to the IDT for Illumina Nextera UD Indexes support page. The IDT for Illumina Nextera UD Indexes incorporate 10 base pair index codes and might require adjustments to set up your sequencing.

Read lengths shorter than 2x250 are likely sufficient when resequencing. However, for de novo assembly, it might be harder to assemble the genome.

Loading Recommendations for Nextera DNA Flex Libraries

Refer to the Prepare Libraries for Sequencing and the Cluster Generation sections in the iSeq 100 Sequencing System Guide.

For Nextera DNA Flex, the loading volume is 20 μl and the loading concentration is 200 pM. The library is automatically denatured into single strands and further diluted onboard the instrument.

For more information, see the Prepare the Flow Cell and Libraries and Cluster Generation sections in the iSeq 100 Sequencing System Guide.

Background Information on PhiX

See the technical bulletin What is the PhiX Control v3 Library and what is its function in Illumina NGS?

Determining the Number of Cycles Required

Refer to the Number of Cycles in a Read section in the iSeq 100 Sequencing System Guide.

Choosing Between Paired-End and Single-Read Sequencing

Paired-end sequencing allows users to sequence both ends of a fragment and generate high-quality, alignable sequence data. Paired-end sequencing facilitates detection of genomic rearrangements and repetitive sequence elements, as well as gene fusions and novel transcripts.

Because paired-end reads are more likely to align to a reference, the quality of the entire data set improves. All Illumina next-generation sequencing (NGS) systems are capable of paired-end sequencing.

Single-read sequencing involves sequencing DNA from only one end and is the simplest way to use Illumina sequencing. By using proprietary reversible terminator chemistry and a novel polymerase, this solution delivers large volumes of high-quality data, rapidly and economically.

For more information, see the Illumina Website and the Dual-Indexed Workflow on a Paired-End Flow Cell (Workflow B) section of the Indexed Sequencing Overview Guide.

Optimizing Cluster Density

Refer to Cluster Optimization Overview Guide and Cluster Density Guidelines on Illumina Systems.

Patterned Flow Cells

Patterned flow cells contain billions of nanowells at fixed locations across both surfaces of the flow cell. The structured organization provides even spacing of sequencing clusters.

For more information, see the Patterned Flow Cell Technology web pagevideo, and technical note.

Analyzing Data for Nextera DNA Flex on the iSeq 100 System

Analyzing NGS Data

Refer to Introduction to Key Concepts in Sequencing Data Analysis webinar for an introductory presentation and discussion on the concepts and general approaches used in analysis of Illumina sequencing data. This webinar is targeted to new to Illumina NGS users and covers the basic analysis approaches used in de novo assembly, RNA sequencing, and SNP finding. Basic concepts in experimental design are also discussed.

Required Equipment

If using Local Run Manager for analysis, equipment and computing requirements are available in the Installation section of the Local Run Manager Software Guide.

Available Public Data on BaseSpace Sequence Hub

The following data are available:

  • MiSeq Sequencing System: Nextera DNA Flex (replicates of E. coli, B. cereus, and R. sphaeroides)
  • MiniSeq Sequencing System: Nextera DNA Flex (replicates of E. coli, B. cereus, and R. sphaeroides)
Comparability of Nextera DNA Flex and Nextera XT in De Novo Assembly Metrics

Refer to the Comparison of de novo assembly metrics section of the Direct Bacterial Colony Sequencing with the Nextera DNA Flex Library Prep Kit application note.

Comparison of Nextera DNA Flex and Nextera XT Whole Genome Coverage

Coverage plots reveal that the Nextera DNA Flex methods, including the direct colony method, all achieve greater uniformity of coverage compared to the Nextera XT DNA method, regardless of which organism is tested or the GC content.

For more information, see the Comparison of whole-genome coverage section of the Direct Bacterial Colony Sequencing with the Nextera DNA Flex Library Prep Kit application note.

Creating a Sample Sheet

You can use the iSeq 100 System Sample Sheet Template to create the sample sheet. A sample sheet is required when sequencing in Manual mode with BaseSpace Sequence Hub Run Monitoring and Storage. Download and edit the sample sheet, and then upload it to the control software during run setup.

For an introduction to run setup, see the Set Up a Sequencing Run (Local Run Manager Mode) or Set Up a Sequencing Run (Manual Mode) sections of the iSeq 100 Sequencing System Guide.

Nextera DNA CD Index Adapter Sequences and IDT for Illumina Nextera UD Indexes

See the Sequences for Nextera Kits section of the Illumina Adapter Sequences Document and the Pooling Guidelines for Nextera Kits section of the Index Adapters Pooling Guide

Index Representation

Good index representation indicates that each library, when multiplexed together with other libraries, receives roughly equal representation of coverage on the flow cell. This translates into more uniform coverage and accuracy of results for a given experiment. 

For more information, see the Index representation and library insert size comparisons section of Direct Bacterial Colony Sequencing With the Nextera DNA Flex Library Prep Kit application note

Local Run Manager

Local Run Manager is a Windows-based software for Illumina benchtop sequencing data, which includes local analysis options and run and user management. The Local Run Manager webinar provides an overview of Local Run Manager and is targeted for new and intermediate users. For more information, view the Local Run Manager support page and the Local Run Manager Overview Online Training Course.

For information on setting up Local Run Manager, see Sequence File FormatsFASTQ Processing Tools for Data Analysis webinar, and Local Run Manager Introduction webinar.

For information on the minimum system requirements for installation, see the Installation section in the Local Run Manager Software Guide.

Using BaseSpace Sequence Hub

BaseSpace Sequence Hub is the Illumina cloud-based sequencing data analysis solution. For information on topics, such as the difference between runs and projects, working with data, and sharing your data, see the BaseSpace Sequence Hub webinar. For more information on using BaseSpace Sequence Hub, see the BaseSpace Sequence Hub support page and the Preparing Runs with BaseSpace Sequence Hub training course.

Analyzing FASTQ Files From iSeq Using BaseSpace Sequence Hub

Refer to the following resources:         

Microbiology Analysis Apps Available Through BaseSpace Sequence Hub

The following analysis apps are available:

  • MetaPhlAn: Metagenomic Phylogenetic Analysis-metagenomics analysis-resulting file sizes are smaller and quicker. MetaPhlAn (Metagenomic Phylogenetic Analysis) is a computational tool for profiling the composition of microbial communities from metagenomic shotgun sequencing data. MetaPhlAn relies on unique clade-specific marker genes identified from reference genomes, allowing orders of magnitude speedups and unambiguous taxonomic assignments.
  • QIIME Preprocessing: QIIME is designed to take users from raw sequencing data generated on the Illumina or other platforms through publication quality graphics and statistics. This includes demultiplexing and quality filtering, OTU picking, taxonomic assignment, and phylogenetic reconstruction, and diversity analyses and visualizations. QIIME has been applied to studies based on billions of sequences from tens of thousands of samples.
  • QIIME Visualizations: QIIME is designed to take users from raw sequencing data generated on the Illumina or other platforms through publication quality graphics and statistics. This includes demultiplexing and quality filtering, OTU picking, taxonomic assignment, phylogenetic reconstruction, and diversity analyses and visualizations. QIIME has been applied to studies based on billions of sequences from tens of thousands of samples.
  • Whole Genome Sequencing (BSSH): The Whole Genome Sequencing App is a rapid alignment, variant calling, and annotation tool for whole-genome sequencing data. The app maps reads using the Isaac aligner [1,2], detects variants (SNVs, small indels, copy number variants, and structural variants), annotates variants, and calculates related metrics.
  • BWA Aligner: Aligns reads to a reference or custom genome - Use if reference genome is not on BS, ie Human genome (mostly for small genomes).
  • Velvet De Novo Assembly: De novo assembly pipeline for bacterial samples using the Velvet assembler. This app has a particular focus on samples sequenced with the Nextera Mate Pair Library Prep kit.
  • Prokka Genome Annotation: Prokka is a software tool to rapidly annotate genes and identify coding sequences in prokaryotic genomes. It is suitable for annotating de novo assemblies of bacteria, but not appropriate for human genomes (or any other eukaryote).
  • SPAdes Genome Assembler: Assembles genomes from paired-end, single, or mate-pair reads. De novo assembler suitable for single-cell and isolate genomes.

Microbiology sequencing (for isolates):

  • GoSeqIt- Bacterial Analysis Pipeline: Takes in assembled genomes from SPAdes or Velvet Assembly apps. Predicts the species, anti-microbial resistance genes, MLST analysis typing, virulence factors, plasmid finder.
  • SRST2- BaseSpace Sequence Hub Lab App: The SRST2 app reports the presence of STs (sequence types) from an MLST database and/or reference genes from a database of sequences for virulence genes, resistance genes, and plasmid replicons.

Third party tools/software (microbiology/isolate):

  • Applied Math Bionumerics: Software suite for bacterial analysis including MLST and SNP typing
  • PubMLST: Public databases for molecular typing and microbial genome diversity
  • RAST: Genome annotation
  • BACWGSTdb: Bacterial Whole Genome Sequence Typing

Additional Resources for Library Prep

Resource Description
Nextera DNA Flex Library Prep Reference Guide Provides comprehensive information on Nextera DNA Flex library prep, including a detailed protocol.
Nextera DNA Flex Library Prep Consumables & Equipment List Interactive list of consumables and equipment used with the Nextera DNA Flex Library Prep kit.
Index Adapters Pooling Guide Provides guidelines for preparing libraries with balanced index combinations for sequencing on Illumina systems.
Illumina Adapter Sequences Document Oligonucleotide (oligo) sequences of Illumina adapters used in Nextera, TruSeq, TruSight, and AmpliSeq for Illumina library prep kits. This information is provided for use with Illumina instruments only.
Nextera DNA Flex Library Prep support page Provides documentation, training resources, frequently asked questions, and information on compatible Illumina products.

Additional Resources for Sequencing and Analysis

Resource Description
iSeq 100 Sequencing System Guide Provides a system overview and instructions for operating and maintaining the iSeq 100 Sequencing System.
iSeq 100 Sequencing System Setup Poster Instructions on installing and setting up the iSeq 100 Sequencing System.
iSeq 100 Sequencing System Site Prep Guide Provides lab specifications and requirements to prepare a site for the iSeq 100 Sequencing System.
Local Run Manager Software Guide  Overview of the Local Run Manager software and instructions for installing analysis modules on the instrument computer.
iSeq 100 Sequencing System support page Provides documentation, training resources, frequently asked questions, and information on compatible Illumina products.