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Guidelines for Custom Target Enrichment Sequencing Projects

The High Throughput Genomics Core Facility supports two options that enable Custom Target Enrichment. These options include the SureSelect XT2 and Haloplex products manufactured by Agilent Technologies. A description of these products and recommendations for preparing samples for exome enrichment or custom target enrichment is described below:

  1. Custom Target Enrichment Kits supported by the High Throughput Genomics Core Facility include the following:
    1. The Agilent SureSelect XT2 Custom Target Enrichment kit recommends an input of 200 to 1000 ng of DNA in a volume of 100 ul. Genomic DNA is mechanically sheared with a Covaris AFA instrument and DNA fragments are selected for an average size of 230 bp. Library preparation with this kit enables pre-enrichment addition of index tags which allows individual libraries to be pooled during an overnight hybridization to a set of biotinylated RNA baits which increases the number of samples that can be simultaneously processed.
    2. The Agilent Haloplex Custom Target Enrichment kit recommends an input of 120 ng of genomic DNA in a volume of 60 ul. Genomic DNA is fragmented by eight unique sets of restriction enzymes (each set containing two enzymes) which results in DNA fragments that vary in size from 100 to 500 bp. Custom enrichment of genomic DNA targets requires a hybridization that ranges from three hours to overnight based on the size of the capture region. Libraries constructed with this kit will have a limited number of DNA ends defined by the combinations of the sixteen restriction enzymes used in the fragmentation reaction.
  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).
  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 SureSelect XT2 kits, however, it is likely to underperform when used with the Haloplex kit due to the restriction enzyme mediated fragmentation method used with this 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.