This resource provides step-by-step guidance through the process of transitioning to Nextera DNA Flex Library Prep on the iSeq 100 System.
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.
The following data sheets provide information on the Nextera DNA Flex Library Prep kit compared with other Illumina DNA library prep kits.
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.
Contact your local sales representative for help calculating the cost per sample.
Nextera DNA Flex supports the following sample types:
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).
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.
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.
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.
The following have been tested using the Nextera DNA Flex Microbial Colony Extraction Demonstrated Protocol Guide:
For information on assessing DNA sample quality, see the Assess DNA Purity section of the Nextera DNA Flex Reference Guide.
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:
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.
For an introduction to using the Nextera DNA Flex Library Prep kit, view the following resources:
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.
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.
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.
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:
The following resources are available:
See above under Initial Considerations.
See the Nextera DNA Library Prep Performance Table in the Nextera DNA Flex Library Prep Kit Data Sheet.
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.
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.
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.
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:
Several factors can impair the performance of the Nextera enzyme:
For additional information, see Nextera XT Library Prep: Tips and Troubleshooting.
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.
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.
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.
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.
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.
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.
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.
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.
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.
For information on on-bead tagmentation, refer to the following resources:
The following resources are available:
For information on SBS technology, view Sequencing: Illumina Technology video or refer to An Introduction to Next-Generation Sequencing Technology.
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.
Documentation and Training Videos can be found on the iSeq 100 Sequencing System support page.
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.
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:
The optimal sequencing depth varies depending on the application you are running and your experimental goals. Refer to the literature for applicable reference studies.
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.
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.
See the technical bulletin What is the PhiX Control v3 Library and what is its function in Illumina NGS?
Refer to the Number of Cycles in a Read section in the iSeq 100 Sequencing System Guide.
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.
Refer to Cluster Optimization Overview Guide and Cluster Density Guidelines on Illumina Systems.
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 page, video, and technical note.
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.
If using Local Run Manager for analysis, equipment and computing requirements are available in the Installation section of the Local Run Manager Software Guide.
The following data are available:
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.
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.
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.
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.
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 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 Formats, FASTQ 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.
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.
Refer to the following resources:
The following analysis apps are available:
Microbiology sequencing (for isolates):
Third party tools/software (microbiology/isolate):
| 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. |
| 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. |