This product was formerly named Nextera DNA Flex.
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Normalization in Illumina DNA Prep happens during tagmentation, hence there is no need to perform bead-based normalization at the end of library prep.
The kit includes a sufficient volume of Sample Purification Beads reagent to process the intended number of samples for the kit. Do not use beads or columns from any other manufacturer with this protocol.
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 their performance which may impact genomic coverage.
No. Illumina DNA Prep chemistry is not compatible with the enrichment chemistry.
Prep is optimized for the use of K2-EDTA and K3-EDTA tubes (manufactured by BD). Other blood tubes may work, but they have not been thoroughly validated.
The following additional tubes have also been tested (1 sample for each tube, tested on the same day as collection).
Store and transport at 2-8°C. Perform lysis reactions within 3 days of blood being collected for optimal results.
This has not been thoroughly validated.
Yes, Illumina DNA Prep can be used on blood spots on cards.
This is dependent on white cell count of the blood sample. A sample with average white cell count (4-7 x 106 leucocytes per mL blood on average for healthy individuals) the estimated amount of DNA in 10µl of blood is 200-600ng.
This is not recommended as it may affect the efficiency of the lysis reaction which will ultimately impact the amount of DNA obtained and therefore the final library yield.
This kit has not been fully validated with blood from a finger stick.
This protocol has been developed to work with the Oragene saliva collection tubes. Saliva samples collected into other tube types may not be stable under the same conditions and therefore the use of other collection tubes is not recommended.
The buccal swab sample type has not been tested with this protocol. Oragene saliva tubes are the only collection method that has been tested.
Samples are stable at room temperature for up to 5 years. Refer to information from DNA Genotek.
The sample may be less stable due the stabilization reagent in the lid of the tube being at a different final concentration in the sample.
Oragene/saliva samples may also be stored at -20°C or -80°C. Samples may undergo at least three freeze-thaw cycles with no evidence of DNA degradation. Refer to information from DNA Genotek.
No, the incubation can be performed at any point prior to beginning the prep. Following the 50°C incubation step the sample is stable at room temperature for up to 5 years.
Samples should be incubated for at least 1 hour but this incubation can also be performed overnight.
This reagent stabilizes the saliva sample.
The amount of bacterial DNA present is variable between different samples and donors. In approximately 80% of the samples that were tested at least 80% of the DNA obtained aligned to the human reference sequence.
The amount of DNA in a saliva sample is very variable between samples and donors. Based on crude Qubit reading from saliva samples, assuming 50% of the DNA is captured during lysis step, the amount of DNA ranges from 34 ng to 2.7 µg.
This is not recommended as it may affect the efficiency of the lysis reaction which will ultimately impact the amount of DNA obtained and therefore the final library yield.
There are 3 safe stopping points in the protocol:
Assess the quality of genomic DNA by running an aliquot of the sample (approximately 10–100 ng) on a 1% agarose gel stained with SYBR Stain. High quality, intact genomic DNA appears as a high molecular weight band (> 10,000 bp) in the absence of a lower molecular weight smear. Low molecular weight smearing can indicate the presence of RNA or degraded DNA.
This kit is optimized for gDNA (dsDNA) and will not work on ssDNA/RNA.
Library yield is not normalized. Reduced diversity/increased duplicates.
Additional PCR cycles can be implemented, as detailed in the user guide.
Although testers have successfully generated libraries from more than 500 ng input DNA, that application is not supported.
Yes. A variety of genomes have been tested and the data remains consistent. A variety of genomes, prepared with Illumina DNA Prep, from bacteria, plants, agriculture, and human can be found on BaseSpace Sequence Hub.
The quality of DNA isolated from FFPE samples can be highly variable. Due to this variability, it is very difficult to reliably predict the quality of a library prepared from FFPE samples using this protocol. Illumina does not support FFPE or degraded DNA as input for this this protocol. Failed libraries originating from this sample type are not eligible for replacement or troubleshooting by Illumina.
Yes. The PCR amplicon must be > 150 bp. Shorter amplicons can be lost during the library cleanup step. Tagmentation cannot add an adapter directly to the distal end of a fragment, so a drop in sequencing coverage of ~50 bp from each distal end is expected. To ensure sufficient coverage of the amplicon target region, design primers to extend beyond the target region by 50 bp per end.
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 less than 100 ng DNA input, we recommend quantification. To quantify input genomic DNA and DNA in the PCR and enriched libraries, use a fluorometric-based method that is specific to 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 Illumina DNA Prep Reference Guide.
1 to 96 samples can be processed at a time through the protocol.
For more information, see the Index Adapter Pooling Guide.
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.
This size-selection step ensures that only fragments of the desired size go into PCR, optimizing the enrichment for the best results.
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 about Sample Purification Bead Size Selection and Best Practices go to the online training sequencing courses to view the online TruSeq Sample Purification Bead Size Selection and Best Practices.
Samples froth because of a detergent used in the library prep. As long as the wash step is included (as described in the protocol), the libraries will not be affected. If desired, gentle pipetting up and down will reduce the amount of froth.
Indel precision and recall is impacted by both library prep and the informatics approach used to call variants. In some cases, you may see improved indel recall and precision by reducing PCR cycles, but this will also result in correspondingly lower yield. If too many PCR cycles are removed, there may not be enough library yield for sequencing.
Yes, but you will need to quantify the library product.
Yes, you can still proceed.
No, if the BLT is dry then you should not proceed.
Let them incubate for a longer period until the beads clear.
Resuspend carefully by pipetting up and down several times, and then proceed with the protocol.
Proceed as long as beads are ‘clearing’ after incubation on the magnet.
Visually inspect and re-clear the supernatant on the magnet if you think you transferred some beads.
Yes, but we do not see added benefit in normalization after beads are saturated.
We have tested frozen reagents up to 4 freeze/thaws of frozen reagents without observing any loss of activity.
It takes 2 to 3 hours from genomic DNA input until libraries are ready to load on the flow cell. The time is dependent on the number of libraries that are prepared.
Unique dual indexes from 2- to 96-plex is supported by the 96-index plate configuration. Refer to the Index Adapters Pooling Guide to get details for unique dual indexing of samples.
IDT for Illumina DNA/RNA UD Indexes uses 10 base pair index codes, whereas Nextera DNA CD Indexes uses 8 base pair index codes. Therefore, you may need to adjust your sequencing setup. For more information, see IDT for Illumina DNA/RNA UD Indexes.
No, because it is a double stranded DNA library and both strands are sequenced.
When using DNA input of 100–500 ng, the library prep normalization features obviate the need to quantify and normalize individual libraries generated within one experiment.
However, you might observe slight variations in the final yields between different library preparation events. To achieve optimal cluster density, pool the libraries with equal volume and quantify the pool.
Because variations between library preparation events and users are possible, individually quantify and normalize the libraries when pooling samples from different experiments.
Use an Agilent Technologies 2100 Bioanalyzer to check the quality and intended size distribution of a tagmented sample, the pre-enriched library, and the post-enriched library. For examples of bioanalyzer traces and library size distributions, see the library prep reference guide. Variation in the Bioanalyzer profiles is expected because it is dependent on the input DNA type.
It is not atypical to see a small bump/shoulder/peak at 1000-3000 bp. The appearance of the peak can vary between DNA samples and does not affect downstream sequencing. We do not know what this peak is and data is unaffected by this peak.
600 bp average fragment length on Bioanalyser (150-1500bp size range).
350 bp median insert size from sequencing.
Actual yield may vary based on sample quality and sample type, typically for good quality DNA sample the expected library yield is greater than 4 ng/ul and within an experiment the CV of library yield is <20%.
The profile of the amplified library product can look different from the example shown depending on the type and quality of input DNA. The use of BLT and a double-sided size selection helps in maintaining the product length. However, inaccurate pipetting during the double-SPRI will cause fragment size variation. Some variation is expected in the profiles of different libraries.
Methods are written and tested by our automation partners to support both 24 & 96 sample kit configurations, including a full plate run with up to 96 reactions. The 96-sample kit is designed to be automation-friendly, including additional volumes necessary for automated processing.
No, automation is only supported by the 24-plex and 96-plex index plates provided by Illumina DNA Prep.
Methods designated "Illumina Qualified" are developed, distributed, and supported by our automation partners. Visit High-Throughput Library Prep Automation to learn more about the partnership program and partners.
In general, Illumina DNA Prep is designed to be compatible with most automated liquid-handling systems.
All Illumina automation partners are developing methods for at least one existing automation platform. Contact the individual partners for more information. To view a list of partners, see High-Throughput Library Prep Automation.
All reagents provide sufficient overage volume to support automation requirements in the 96-sample kit configuration. Additionally, the 96-sample kit configuration should support at least two runs of 48 samples each (2 runs x 48 samples).
We recommend utilizing Illumina Qualified* methods from our partners, benefits include:
*Illumina Qualified indicates that libraries prepared with this method have been shown to perform comparably to those prepared manually.
See the Index Adapters Pooling Guide to make sure your sequencing set up is appropriate for your sequencing platform.
Yes, but the libraries will need to quantified before pooling. If input DNA was at least 100 ng, libraries from each independent experiment may be pooled and quantified together prior to pooling with the second experiment. It is not recommended to pool all samples from independent experiments prior to quantification due to variability observed from user to user.
Yes, there are example data sets in BaseSpace Public Data.
If you are using the following software products to sequence Illumina DNA Prep libraries, make sure that you use the indicated version. For information on which software you need, see Illumina DNA Prep Compatible Products.
A paired-end flow cell from any Illumina sequencing system can be used to sequence these libraries.
This is a normal characteristic of the tagmented libraries and relates to the sequence context at the tagmentation site.
During run creation, select the Show advanced module settings option. From here select the Add Custom Setting option. For read 1 adapter trimming, enter Adapter as the option name and the appropriate sequence. For Illumina DNA Prep the sequence is CTGTCTCTTATACACATCT.
Download the Illumina DNA Prep definition file from the Product Files page of the Illumina DNA Prep support site to a known location such as the desktop. Open the Local Run Manager application and select the Gear (System Settings) icon. From here, select Library Prep Kits and select the Add Library Prep Kit option. Navigate to the location of the template file and add the file. The new kit will now appear under the Library Prep Kit dropdown menu when creating a run.
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