Iowa Institute of Human Genetics

  • Genome Sequencing

    Library Prep Workflows

    DNA-Seq Workflow

    The IIHG Genomics Division provides genomic DNA sequencing that includes sample QC, library creation and sequencing. The IIHG Genomics Division supports the use of the TruSeq DNA PCR-Free Sample Preparation Kits, but will work with other protocol as input amount and number of samples warrant consideration of a different protocol.

    Sample Submission

    At least 2 ug of DNA diluted in 20 ul nuclease free H2O is requested. It is recommended researchers check genomic DNA by running the sample on a 1% agarose gel. Intact genomic DNA should appear as a high molecular weight band (> 10,000 bp) with no lower molecular weight smear. If possible, please provide a picture of the gel when submitting your sample. RNase I treatment is highly recommended.

    Quality Control

    We quantify the samples using fluorimetry. For a sample to pass QC, there must be ≥ 1ug. 

    Library Creation

    Steps include DNA shearing using the Covaris, fragment purification and end polishing, and ligation to indexed (barcoded) adaptors. The sequencing library is then size selected (if desired), size distribution validated using the Agilent Bioanalyzer, and quantified using the qPCR-based KAPA Assay.  

    Sequencing

    Indexed libraries are normalized, pooled (depending on number of reads requested), clustered on a flow cell using the cBOT cluster station, and loaded onto the instrument for sequencing.

    RNA-Seq Workflow

    The IIHG Genomics Division provides RNA-Seq which includes sample QC, library creation, and sequencing to your preferred read depth. The division supports the Illumina RNA-Seq stranded and non-stranded protocols as well as the NuGen Ovation Ultralow library systems for low quantity samples.  

    Samples

    Please submit at least 2 ug total RNA for the Illumina RNA-Seq workflows or 10 ng for the NuGen workflow. The preferred RNA extraction method is via QIAGEN RNeasy kit. Organic RNA extraction methods, such as phenol or Trizol, should be cleaned up thoroughly. Organic carryover can inhibit the enzymatic reactions used in Illumina library preparation and can increase the risk of failure of library generation. 

    Quality Control

    Samples are quantified using fluorimetry and RNA quality is assessed using the Agilent BioAnalyzer 2100, generating an RNA Integrity Number (RIN). For a sample to pass QC, mass must be ≥ 1 ug, with a RIN > 8 to enter the Illumina RNA-Seq library preparation workflows. At least 0.5 ng are requested to enter the NuGen RNA-Seq workflow. 

    Library Creation

    Depending on the library protocol steps include oligo-dT purification of polyadenylated RNA, and reverse transcription to create cDNA. The cDNA is fragmented, blunt-ended, and ligated to indexed (barcoded) adaptors. The library is size selected (in the case of paired-end RNA-Seq), size distribution validated using capillary electrophoresis, and quantified using fluorometry and via qPCR. 

    Sequencing

    Indexed libraries are normalized, pooled (depending on number of reads requested), clustered on a flow cell, and loaded onto the instrument for sequencing. 

    Target Capture Using Agilent SureSelect

    The IIHG Genomics Division provides target capture for whole exome and custom targeted panel sequencing that includes sample QC and library creation.

    Sample Submission

    At least 3 ug of DNA, as measured by fluorometry, diluted in nuclease free water is requested. It is recommended researchers check genomic DNA by running the sample on a 1% agarose gel. Intact genomic DNA should appear as a high molecular weight band (> 10,000 bp) with no lower molecular weight smear. If possible, please provide a picture of the gel when submitting your sample. RNase I treatment is highly recommended.

    Quality Control

    We quantify the samples using fluorometry. For a sample to pass QC, there must be ≥ 3 ug. The gel image should indicate high MW DNA and the A260/280 should be greater than 1.8. 

    Library Creation

    Libraries are prepared by shearing the DNA using the Covaris, fragment clean-up using AMPure, end polishing, and ligation of adaptors. Following normalization, the libraries are hybridized to the capture baits, and a barcode is added to each captured library via PCR amplification with a barcoded primer. The barcoded capture libraries are quantified, and pooled for sequencing.

    Sequencing

    Indexed libraries are normalized, pooled (depending on number of reads requested), clustered on a flow cell, and loaded onto the instrument for sequencing. Three whole exome capture libraries are typically loaded into each lane of a HiSeq to give at least 10x coverage of greater than 90% of the exome target.