Library Prep Workflows

(First time users should contact the Genome Sequencing Service to obtain best practices for sample preparation prior to sample submission)


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 KAPA Hyper Prep Kits, but will work with other protocols as input amount and number of samples warrant consideration of a different protocol.

Sample Submission

At least 2 ug of DNA diluted in 50 ul 10 mM Tris, pH 8 (Qiagen EB buffer is fine) in 1.5 ul low-bind microfuge tubes are 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.

Samples

Please submit at least 2 ug total RNA for the Illumina RNA-Seq workflows. 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.

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. 


Whole Exome and Custom Target Sequencing

The IIHG Genomics Division provides target capture for whole exome and custom targeted panel sequencing that includes sample QC and library creation. First time providers of samples for Target Capture are strongly encouraged to first contact the Genome Sequencing group prior to sample preparation to obtain best practices for sample submission.

Preparing and submitting samples for Whole Exome Sequencing

Below are our recommended or “best practices” workflow to prepare samples for whole exome sequencing 

RNase I treatment is highly recommended.

QC of samples prior to submission is essential (Mix well before each measurement) and is best performed as follows:

Check genomic DNA by running 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.

Measure the samples on the Nanodrop to determine concentrations (ng/ul), and purity (260/280, 260/230). The 260/280 values should be ~1.8-2.0 and the 260/230 values should be above 2.0 (or at the very least above 1.0). If the concentrations are above 100ng/ul, dilute at least 4ug to around 60 ng/ul for measuring on the Qubit.

Measure samples (under 100ng/ul based on Nanodrop) on the Qubit with the DNA High Sensitivity kit. Use these results to aliquot 3 ug, then add EB to a total volume of 130ul.

We would welcome the opportunity to review your QA/QC results before you submit your samples.  This includes a gel image and an Excel file that includes: Nanodrop (ng/ul, 260/280, 260/230); Dilution factor (after Nanodrop and before Qubit measurement); Qubit HS (ng/ul); The volume that was aliquoted and the volume of EB that was added). 

Sample Submission: Please submit 3 ug of DNA as measured with a Qubit DNA HS kit diluted in 130 uL 10 mM Tris, pH 8 (e.g., Qiagen EB buffer).  If you are able, please double this amount (6ug in 260ul). Samples should be submitted in 1.5ml low-bind tubes labelled with simple numbers.   Samples should be delivered to 116 EMRB.


Contact Information

116 EMRB
Phone: (319) 335-6736 
Fax: (319) 335-6737 
dna-genomeseq@uiowa.edu

For information about downstream bioinformatics support, contact the IIHG Bioinformatics Division (319) 335-6717.