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Guidelines for Exome Enrichment Sequencing Projects

The High Throughput Genomics Core Facility supports two options for Exome Enrichment Sequencing Projects. These options include the SureSelect XT and the SureSelect QXT enrichment products manufactured by Agilent Technologies. A description of these products and recommendations for preparing samples for exome enrichment is described below:

  1. Exome Enrichment kits supported by the High Throughput Genomics Core Facility include the following:
    1. The Agilent SureSelect XT Human All Exon v5 plus UTR enrichment kit recommends an input of 200-3000 ng of DNA (in a volume of 100 ul) to construct a library for exome enrichment. Genomic DNA is mechanically sheared with a Covaris AFA instrument and DNA fragments are selected for an average size of 230 bp. Enrichment of this library requires an overnight hybridization to a set of biotinylated RNA baits of an average length of 120 nucleotides. The SureSelect Human All Exon v5 plus UTR kit targets 71 MB of genomic sequence representing 335,765 exons in 21,058 genes and corresponding UTRs. All libraries constructed with this kit include an index tag that is added following enrichment and that enables multiplex sequencing.
    2. The Agilent SureSelect XT Clinical Research Exome enrichment kit recommends an input of 200-3000 ng of DNA (in a volume of 100 ul) to construct a library for exome enrichment. Genomic DNA is mechanically sheared with a Covaris AFA instrument and DNA fragments are selected for an average size of 230 bp. Enrichment of this library requires an overnight hybridization to a set of biotinylated RNA baits of an average length of 120 nucleotides. The SureSelect Clinical Research Exome kit targets 54 MB of genomic sequence and has been optimized for enhanced coverage in disease-associated regions as defined by databases such as Online Mendelian Inheritance In Man (OMIM), Human Genome Mutation Database (HGMD) and NCBI's ClinVar. All libraries constructed with this kit include an index tag that is added following enrichment and that enables multiplex sequencing.
    3. The Agilent SureSelect QXT All Exon v5 plus UTR enrichment kit enables exome enrichment from a quantity of 50 ng of DNA (column purified and EDTA-free) in a 10 ul volume. Genomic DNA is fragmented by a transposase activity that results in DNA fragments that range from 100-500 bp with an average size of approximately 230 bp. Following library construction, exome enrichment requires a 90 minute hybridization to biotinylated RNA baits and therefore, library construction and enrichment can be completed in a single day. The SureSelect Human All Exon v5 plus UTR kit targets 71 MB of genomic sequence representing 335,765 exons in 21,058 genes and corresponding UTRs. All libraries constructed with this kit include an index tag that is added following enrichment and that enables multiplex sequencing.
    4. Additional SureSelect Exome Capture kits are available which enable enrichment of coding sequence for mouse, canine, bovine and zebrafish. Please inquire with the core facility if you are interested in using one of these products.
  2. Purification of Genomic DNA: Genomic DNA should be purified using a column-based purification protocol such as the Qiagen DNeasy Blood and Tissue kit (cat#69504), or one of the DNA purification kits from Zymo Research (Quick-gDNA MiniPrep, Quick-gDNA Blood MiniPrep, or ZR Genomic DNA-Tissue MiniPrep). We recommend elution of the DNA in 10mM Tris pH 8 as an alternative to using the Qiagen elution buffer (Buffer AE) or the Zymo Research Elution Buffer, both which contains EDTA. The omission of EDTA from the elution buffer is required for the use of SureSelect QXT system as the transposase activity of the QXT kit will inefficiently fragment genomic DNA in the presence of EDTA.
  3. Purification of DNA from FFPE Tissues: Genomic DNA from FFPE tissues should be purified using a column-based purification protocol such as the Qiagen DNA FFPE Tissue Kit (cat#56404), the Zymo Research ZR FFPE DNA MiniPrep, or the Ambion RecoverAll Total Nucleic Acid Isolation Kit for FFPE (cat#AM1975). DNA extracted from FFPE tissues can only be used with the SureSelect XT kit as it will likely underperform when used with a transposase-based fragmentation method such as that used with the QXT kit.
  4. Avoid Organic Extraction Methods: We recommend that you avoid organic extraction methods (such as phenol or Trizol) or other methods that include an alcohol precipitation step. The quality of DNA purified by these protocols tends to be lower due to co-precipitation of contaminants. Furthermore organic carryover can inhibit the enzymatic reactions used in library preparation.
  5. Assessment of DNA Concentration: The concentration of genomic DNA can be determined using the Qubit dsDNA BR assay (Invitrogen cat#Q32850) or the Qubit dsDNA HS assay (Invitrogen cat#32851). 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/ul to 1000 ng/ul. In contrast, a NanoDrop measurement often exaggerates the concentration of purified DNA as any RNA that co-purifies with the DNA will contribute to the absorbance measurement at 260 nm.
  6. Assessment of DNA Quality: The quality of genomic DNA can be assessed by running an aliquot of the sample (approximately 10-100 ng) on a 1% agarose gel stained with SYBR Safe DNA Gel Stain (Invitrogen cat#S33102). High quality, intact genomic DNA should appear as a high molecular weight band (>10,000 bp) in the absence of a lower molecular weight smear. Low molecular weight smearing can be indicative of the presence of RNA.
  7. Library QC: Sequencing libraries will be validated by running an aliquot on an Agilent 2200 TapeStation and by defining the molarity of the library using a qPCR assay (Kapa Biosystems Library Quantification Kit for Illumina). After normalizing library concentrations following the completion of these two assays, libraries can be pooled in preparation for sequencing.