Questions & Answers

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Input

  • What is the necessary length of input material (i.e., if starting from DNA that is already fragmented, amplicons, etc)?

    Illumina recommends >300bp to ensure even coverage across the length of the DNA fragment. An expected drop off in sequencing coverage about 50bp from each distal end of a fragment may be seen. This is because the tagmentation reaction cannot add an adapter right at the distal end of a fragment. For PCR amplicon sequencing this can be easily averted by simply designing your amplicons to be ~100 bases larger than the desired insert to be sequenced.

Protocol

  • How do I check the quality of my library?

    Illumina recommends the BioAnalyzer. Please see the Nextera DNA Sample Preparation Guide for more specific details and example traces.

  • Is DNA fragmentation (i.e., with Covaris) necessary?

    No. The tagmentation step in the Nextera protocol eliminates the need for mechanical fragmentation/shearing.

  • How will I know what are the best index combinations for my sample prep?

    The Illumina Experiment Manager (IEM) will notify you if improper combinations are used when creating a sample sheet for use with CASAVA, so it is highly recommended to create your sample sheet prior to performing sample prep/pooling. There are also low plexity pooling guidelines in the Nextera, TruSeq DNA, and TruSeq RNA sample preparation guides. Always pool samples with valid index combinations to avoid image registration failures.

  • What is the library size distribution for Nextera libraries?

    The Illumina Nextera DNA Sample Preparation kit produces libraries with a broad size range distribution, typically between 300-1000bp. Please refer to the Nextera DNA Sample Preparation Guide for more specific details.

  • What peripheral equipment is needed to perform the Illumina Nextera sample prep?

    A standard thermocycler is required to perform PCR.

  • Are clean‐ups/size selection steps necessary?

    The protocol includes one Zymo cleanup step (following "tagmentation") and one Ampure XP size selection step (following limited cycle PCR). The final library should have a median insert size of ~250-300 to support long paired end 2x150 read lengths on the MiSeq system.

Library Evaluation

  • What quantitation methods does Illumina recommend for final library quantitation?

    Double Stranded DNA specific dyes, such as QuBit or picogreen.

  • Can data from Illumina Nextera libraries be directly compared to legacy Nextera kits?

    The Illumina Nextera kit has been optimized and performances between kits may be different. For example, the new Illumina Nextera DNA Sample Preparation kits exhibit improved GC performance compared to the legacy kits.

  • How is the Illumina Nextera kit different from the legacy Nextera kit from Epicentre?

    The Illumina Nextera kit has improved master mixes resulting in an easier protocol with less tubes. Additionally, there is an optimized PCR that does not require the purchase of any additional components and an optimized tagmentation reaction. Finally, the new dual indexing strategy allows 96 indices per lane and samples can be sequenced on all Illumina sequencing platforms.

  • Are the index sequences for the first 7 cycles of index 1 the same as current TruSeq Kits, TruSeq Custom Amplicon Kits, etc.?

    No. For specific index sequences, please refer to the Illumina Experiment Manager (IEM) or the Illumina Nextera DNA Sample Preparation Guide.

  • What quantitation methods does Illumina recommend for starting material?

    Double Stranded DNA specific dyes, such as QuBit or picogreen.

  • Can Illumina Nextera DNA Sample Preparation kits be used for methylation studies?

    No, because the adaptors are not methylated.

Cluster Generation

  • Can Nextera dual-indexed libraries be combined with other library types on the same flow cell?

    Yes. If you are sequencing combined single-indexed and dual-indexed libraries on the HiSeq or GA, use sequencing primers provided in the TruSeq Dual Index Sequencing Primer Box (Single Read, Catalog # FC-121-1003 or Paired End, Catalog # PE-121-1003). Primers provided in this kit are compatible with all library types. Single-indexed libraries and dual-indexed libraries need to be loaded onto separate lanes of the flow cell. For more information, see Sequencing Mixed Libraries on a HiSeq or GA Flow Cell.

Sequencing

  • Are there any in-line controls in the sample prep?

    No.

  • How long is each Index Read?

    Both Index 1 (i7) and Index 2 (i5) are 8 bp in length.

  • How many uses can you obtain from the index kits?

    The index kits are good for four uses. The 96-index kit has enough reagents for 384 samples; the 24-index kit has enough reagents for 96 samples. Additional caps are included for the tubes to replace after each use in order to prevent cross-contamination.

  • Will the new Nextera dual-indexing support single-indexing as well?

    Yes. 12-plex single-index runs are supported on the HiSeq/GA using i7 indexes only. GA support for Nextera will be available in November 2011 when the latest version of SCS/RTA is available. Please see the Nextera DNA Sample Preparation Guide for more information on pooling recommendations for samples less than the full kit size.

  • How does dual indexing work?

    For paired-end flow cells, dual indexing now requires 23 additional cycles of sequencing - eight cycles for the Index 1 (i7) Read, eight cycles for the Index 2 (i5) Read, plus seven non-imaging, chemistry-only cycles at the beginning of the Index 2 (i5) Read. For single-read flow cells, dual indexing only requires 16 additional cycles of sequencing - eight cycles for the Index 1 (i7) Read and eight cycles for the Index 2 (i5) Read.

  • What level of multiplexing is supported on the GA with Nextera? When will this be available?

    There will be 96 available barcodes using 12x8 indices, combining 12 Index 1(i7) with 8 Index 2 (i5) indices. Multiplexing on GA is not supported until updated SCS/RTA software is available.

  • How do Nextera DNA Sample Preparation Kits work?

    Nextera sample preparation kits are used to prepare DNA samples for next‐generation sequencing. These kits use in vitro transposition to prepare sequencer‐ready libraries from genomic DNA for all Illumina sequencing platforms. The technology simultaneously fragments and tags DNA in a single tube reaction and this process is referred to as "tagmentation".  The protocol takes approximately two hours for a 24 sample prep, with ~60 minutes of hands-on time, and requires 50 ng of starting DNA.

  • Is it a problem to run low numbers of indices/pooled samples? What will happen if I run improper index combinations?

    If there is no signal in one of the color channels of the index read, the image registration might fail and no base will be called from that cycle. If no base is called, the index read may not be able to be matched to the sequence specified in the sample sheet, and then samples will not be able to be demultiplexed.

  • What level of multiplexing is supported on the MiSeq with Nextera?

    There are 96 available barcodes using 12x8 indices, combining 12 Index 1(i7) with 8 Index 2 (i5) indices.

  • Is there a minimum number of indices I need to use?

    For dual indexing, at least 2 barcodes/indices need to be present for each Index Read. For less than 6 samples, Illumina recommends using single indexing. Please refer to the pooling guidelines in the Nextera DNA Sample Preparation Guide.

  • What Illumina sequencing platforms are compatible with Nextera?

    Nextera is compatible with all Illumina sequencing platforms: MiSeq , HiSeq 2000, HiSeq 1000, HiScanSQ , and GAIIx.

  • Do I need to make full 96-plex or 24-plex pools?

    No, lower levels of multiplexing are possible. Please see the Nextera DNA Sample Preparation Guide for more information on pooling recommendations for samples less than the full kit size.

  • What level of multiplexing is supported on the HiSeq with Nextera?

    There are 96 available barcodes using 12x8 indices, combining 12 Index 1(i7) with 8 Index 2 (i5) indices.

  • Can I perform Single Read runs and still get both index sequences?

    Yes. Please select the appropriate workflow based on the flow cell type (Single Read or Paired End) when starting your run in HCS.

  • What applications are suitable for Nextera?

    Nextera library preparation is suitable for genomic DNA applications where speed is paramount, input material is limiting (e.g., less than 100 ng DNA), where the sample prep throughput is a bottleneck, or when non-mechanical fragmentation is required. For example, Nextera is ideally suited to small genome sequencing with MiSeq.

  • Can I run TruSeq HT libraries with Nextera libraries on the same lane or same flow cell? Are the indexes the same or different?

    Illumina does not support, and strongly advises against, running libraries prepared with different library prep kits in the same lane of a flow cell. Running libraries prepared by different library prep kits in different lanes of the same flow cell or spiking in Illumina PhiX control library in the same lane as any user-prepared libraries is supported.

    If different library types are run in different lanes, dual index recipes and the Dual Index Primer Box are required. The indexes for TruSeq HT and Nextera are unique and not shared.

Analysis

  • What software is required/available to support Nextera dual-indexing? Which versions?

    Software Component/version:

    • HCS 1.5/RTA 1.13/SAV 1.8.4
    • SCS 2.10/RTA 1.13/SAV 1.8.4
    • MiSeq Reporter 1.1
    • CASAVA 1.8.2
    • Illumina Experiment Manager 1.0

    Please note that SCS 2.10/RTA 1.13 for GA is scheduled to be available November 2011.

  • What is Adapter Trimming?

    Shorter inserts may lead to sequencing into the adapter, and this feature helps filter out adapter sequence from the final sequence data. Please select this option when creating your sample sheet in IEM for use with MiSeq as the MiSeq Reporter (MSR) analysis software will automatically trim the adapter sequence. For all other run types, this option can be used with the appropriate commands in CASAVA 1.8.2.

  • Is the sample sheet/library sheet optional or mandatory?

    For MiSeq runs, a sample sheet is required at the start of the run to enable analysis. For HiSeq/HiScanSQ/GA runs, creating and loading this sample sheet at the start of the run is optional, but is highly recommended in order to view data in the new indexing tab of SAV during the run. If you do not load a sample sheet at the start of a run in HCS, you will not be able to view indexing data in SAV. Additionally it is recommended to create the sample sheet in the Illumina Experiment Manager (IEM) prior to performing sample prep in order to confirm appropriate index combinations.

  • Would there be issues demultiplexing current TruSeq libraries if a dual index run was done?

    No. You must specify the correct –use-bases-mask and use the appropriate sample sheet to enable demultiplexing. For more information, see the CASAVA User Guide for details on sample sheets and demultiplexing commands.