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Guidelines for DNA Sequencing Projects

The Illumina HiSeq provides a cost effective solution to sequence entire genomes. We support library construction for genomic DNA resequencing projects using four standardized protocols in addition to options for user defined customized size selection or no size selection. We further support short insert libraries (180 bp) which are routinely used for genome assembly projects using ALLPATHS-LG software. Short insert libraries are described under the Genome Assembly Header. Options for DNA resequencing projects are described below:
  1. DNA Sequencing Library Prep kits supported by the High Throughput Genomics Core Facility include the following:
    1. The Illumina TruSeq DNA PCR-Free Sample Prep (350 bp mean insert size) protocol recommends an input of 1000 ng of DNA (in a volume of 50 ul) to construct a library with an average insert size of 350 bp. Libraries are constructed in the absence of pcr to minimize bias that may be introduced by pcr amplification.
    2. The Illumina TruSeq DNA PCR-Free Sample Prep (550 bp mean insert size) protocol recommends an input of 2000 ng (in a volume of 50 ul) of DNA to construct a library with an average insert size of 550 bp. Libraries are constructed in the absence of pcr to minimize bias that may be introduced by pcr amplification.
    3. The Illumina TruSeq Nano DNA Sample Prep (350 bp mean insert size) protocol recommends an input of 100 ng of DNA (in a volume of 50 ul) to construct a library with an average insert size of 350 bp. Eight cycles of pcr are required to complete construction of the library.
    4. The Illumina TruSeq Nano DNA Sample Prep (550 bp mean insert size) protocol recommends an input of 200 ng of DNA (in a volume of 50 ul) to construct a library with an average insert size of 550 bp. Eight cycles of pcr are required to complete construction of the library.
    5. The Illumina TruSeq Nano DNA Sample Prep (no size selection) protocol has been customized to omit a size selection step so that library prep can be performed with a lower quantity of DNA input. We recommend a minimum of 10 ng of DNA (in a volume of 50 ul) to construct a library with no size selection and the average insert size will range from 100-600 bp. Eight cycles of pcr are required to complete construction of the library.
    6. The Illumina TruSeq Nano DNA Sample Prep (custom size selection) protocol requires an input of 200-500 ng of DNA in a volume of 50 ul. Following pcr amplification of the library (8 cycles), size selection is performed by resolving the library on a 2% agarose gel and excising a fragment that represents the target insert size.
  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).
  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.