Nextera DNA Exome FAQs

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  • Input

  • 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, 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 Exome Reference Guide.

    Although the Nextera DNA Exome Enzyme is optimized to tolerate a level of DNA input variability, 50 ng of input genomic DNA is the target for optimum data. It is important to quantify the input genomic DNA accurately to generate a high-quality library of the correct size. Use a fluorometric-based quantification method for the input genomic DNA.

    For more information, see the DNA Input Recommendations section of the Nextera DNA Exome Reference Guide.

    With the Nextera DNA Exome 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 this protocol. This does not mean that FFPE samples cannot be attempted, but that failed libraries originating from this sample type are not eligible for replacement or troubleshooting by Illumina.

  • Library Evaluation

  • 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. 

  • Analysis

  • 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.