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.
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 (eg, QuantiFluor or PicoGreen).
Use the Qubit dsDNA BR Assay or the Qubit dsDNA HS assay to determine the concentration of gDNA. These assays use a fluorescent dye that is highly selective for double-stranded DNA over RNA. It 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 detailed input recommendations, see the TruSeq Rapid Exome Reference Guide.
With the enzyme TDE2, the effects of adding a range of DNA are minimal. The protocol is optimized for 50 ng input DNA, and this amount is recommended. However, the protocol has been validated to have similar final analysis results from 30–75 ng DNA input.
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 protocol. FFPE samples can be attempted using the protocol, but failed libraries originating from this sample type are not eligible for replacement or troubleshooting by Illumina.
No. The Nextera DNA Exome Enrichment reagents and protocol have been optimized to be used together. The kits include all components for both library prep and Rapid Capture. Each kit contains components optimized specifically for the use in that protocol. Deviation from the protocol with reagents from other kits can lead to protocol underperformance or failure.
This protocol has been optimized and validated using the microheating system specified in the library prep reference guide. Comparable performance is not guaranteed when using alternate equipment.
Overloading the enrichment reaction with too much library can lead to an increase in off-target reads. Conversely, loading too little library in the enrichment reaction can lead to lower library diversity and an increase in duplicate sequencing reads.
Quantify the enriched library using a fluorometric-based method specifically for double-stranded DNA, such as QuantiFluor or PicoGreen. For more information, see the library prep reference guide.
You can also use qPCR to quantify the final enriched library. For more information, see the Sequencing Library qPCR Quantification Guide.
Quantify the tagmented library using a fluorometric-based method specifically for double-stranded DNA, such as QuantiFluor or PicoGreen. For more information, see the reference guide.
A thermal cycler leads to better consistency and more robust results across samples with a plate when compared to using a heat block for hybridization. By using a thermal cycler at this step, the 100°C heated lid helps to prevent large sample evaporation.
This kit include all the reagents needed for generating complete tagmented and enriched libraries ready for sequencing. The kits also include magnetic SPRI beads for sample clean-up steps. For more information, see the Kit Contents section of the library prep reference guide.
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.
There are six safe stopping points in the protocol. The safe stopping points are after the following steps:
For storage details, see the reference guide.
TruSeq Rapid Exome is an optimized enrichment workflow that offers tolerance to input DNA variability and shortened hybridization times.
The TruSeq Rapid Exome kit uses an improved tagmentation enzyme. It demonstrates higher enrichment and coverage with improved accuracy of variant calls and Q-scores.
You can prepare one sample at a time. However:
1–96 samples can be processed at a time through the protocol. When processing fewer than the maximum number of samples per kit, store the aliquots of reagents for subsequent use instead of repeated thawing and freezing of the same reagent tubes.
The coding exome (45 Mb) uses 80 mer oligos.
A full list of user supplied items can be found in the Consumables and Equipment section of the library prep reference guide.
For more information, see the Index Adapter Pooling Guide.
No, there is no range of hybridization times. The protocol has been optimized for 30 minutes for each hybridization
This size-selection step ensures that only fragments of the desired size go into PCR, optimizing the enrichment for the best results.
The tube protocol processes samples in Eppendorf tubes and is recommended for processing ≤ 16 samples pre-enrichment. The plate protocol uses a 96-well plate and is recommended for processing > 16 samples pre-enrichment.
The tube and plate protocols require different consumables, magnetic stands, and incubation equipment. Expect equivalent results from either option. However, the plate option can yield more consistent results between samples.
During enrichment, C to A mutations can occur in the DNA. The oxo Q-score meaures the rate of these mutations.
The protocol uses a proprietary buffer to minimalize these mutations.
The profile of the pre-enrichment library product can look different from the example shown depending on the type and quality of input DNA. Sometimes a larger molecular weight peak is present. This peak can be variable in size. However, it has minimal to no effect on the final exome metric output and you can proceed with the protocol. This peak is most often a result of the amplification step of the protocol.
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.
The libraries generally range from ~200–500 bp, with the main peak at ~300–350 bp.
All paired-end cluster kits are compatible with TruSeq Rapid Exome libraries. If preparing these libraries for cluster generation for a HiSeq system, order the TruSeq Dual Index Sequencing Primer kit (catalog # PE-121-1003). Use the Read 1 sequencing primer (HP10) in place of the existing Read 1 sequencing primer (HP6) in the cBot plate.
Yes, there are example data sets in BaseSpace Public Data.
The MiSeq System provides sufficient output to run a single sample per MiSeq run. However, the HiSeq 2500 System is the most cost effective and highest throughput platform for exome sequencing.
The TruSeq Dual Index Sequencing Primer Kit (catalog #PE-121-1003) is required for sequencing on the HiSeq 2500 or HiSeq 2000 using TruSeq v3 chemistry. The box supports one run and contains the required primers for dual-indexed sequencing (HP10, HP11, and HP12).
The dual-index primers are included with all other reagent kits and do not need to be purchased separately.
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.
The kits include 12 different Index 1 (i7) adapters (N701–N712) and up to eight different Index 2 (i5) adapters (E501–E508), depending on the kit. The libraries use the same dual 8 bp indexes as other Nextera-based kits.
For more information on dual-indexing and pooling, see the Index Adapters Pooling Guide.
Yes. All Nextera-based libraries use the same sequencing and index primers (HP10, HP11, HP12) and can be loaded in different lanes of the same flow cell for sequencing.
Manifest files list genomic regions and coordinates targeted for enrichment with the library prep kit. Data analysis requires manifest files for alignment and variant calling in targeted regions. Each product has a _targeted regions and _probe manifest file.
Download manifest files from the Product Files page for the library prep kit.
Human UCSC version hg19, which is the same as Genome Reference Consortium build 37 (GRCh37).
Yes. Download the BED files from the Product Files page for your product.
Analyze HiSeq data using the HiSeq Analysis Software enrichment workflow for HiSeq data. Analyze MiSeq data using MiSeq Reporter. See the HiSeq Analysis Software or MiSeq Reporter support pages for more information.
Alternately, Illumina sequence base call output files (*.bcl) can be demultiplexed and converted to FASTQ format using the bcl2fastq converter software. The files can then be used for analysis with other third party software packages (eg, BWA and GATK). If the user is analyzing with the use of basespace, we recommend the use of BWA or Isaac enrichment analysis tools. If any subsampling is required, use the FASTQ Toolkit.