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Library Prep Workflows

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


RNA-Seq Workflow

The IIHG Genomics Division provides RNA-Seq which includes sample QC, review of QC, library creation, and sequencing to your preferred read depth. The division supports the Illumina TruSeq stranded mRNA-Seq and Total RNA-Seq using ribozero protocols, but will consider other library preparation protocols as input amount and number of samples warrant.  

Sample Submission

For RNA-Seq workflows, please submit 20 ul of total RNA at a concentration of 75-350 ng/ul as measured by a Nanodrop, Qubit or similar.  

DNase I treatment and removal is required.  The division will perform the subsequent QA/QC steps at a cost of $18/per sample.  Alternatively, investigators may choose to perform their own QA/QC (see Alternative RNA-Seq Sample Submission Workflows).

A suggested method for RNA extraction method is via QIAGEN RNeasey kit with on-column DNase treatment/removal.  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

The Genomics Division performs the following QC on RNA Samples:  

  1. Samples are quantified using the Trinean DropSense 16.  The instrument also provides information about possible contaminants in the sample that may impact library preparation.  
  2. RNA quality is assessed using the Agilent BioAnalyzer which generates an RNA integrity Number (RIN).  For a sample to enter the mRNA-Seq workflow, it should have a RIN > 8.  

If your samples don't pass QC please contact us.  It might still be possible to sequence them and we might have suggestions for you.  

Library Creation

Depending on the library, protocol steps include either oligo-dT purification of polyadenylated RNA (mRNA-Seq) or ribosomal-RNA depletion (Total RNA-Seq), followed by reverse transcription to create cDNA. Additional steps include fragment purification, end polishing, and ligation to indexed (barcoded) adaptors. The size distribution of the library is validated using the Agilent Bioanalyzer, and the quantification for sequencing is done using the qPCR-based assay.   

Sequencing

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


DNA-Seq Workflow

The IIHG Genomics Division provides genomic DNA sequencing (e.g, whole genome and amplicons) that includes review of sample QC, library creation and sequencing. The IIHG Genomics Division supports the use of KAPA Hyper Prep Kits, but will consider other protocols as input amount and number of samples warrant.

Sample Submission

At least 1.2 ug of DNA, as measured by Qubit, diluted in 60 ul 10 mM Tris, pH 8 (Qiagen EB buffer) in 1.5 ul low-bind microfuge tubes are requested.  If you are able, please double this amount (2.4ug in 120ul). RNase I treatment of your DNA is highly recommended.

Quality Control

  1. 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 smearing in the lower MW regions.
  2. Measure genomic DNA on the Nanodrop to evaluate purity. For a sample to pass QC, 260/280 ~1.8, and 260/230 above 2.0 (or at least above 1.0)
  3. Measure genomic DNA using fluorimetry (e.g. Qubit) to get an accurate sample concentration.  For a sample to pass QC, there must be ≥ 1ug (by fluorimetry).

Prior to sample submission, please provide a gel image, fluorimetry results (ng/ul), and Nanodrop results (260/280, 260/230). If your samples don't pass QC please contact to us. It might still be possible to sequence them and we might have suggestions for you.

Library Creation

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

Sequencing

Indexed libraries are 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 review of sample QC,  library creation and sequencing.  The IIHG Genomics Division supports the use of Agilent SureSelect XT kits.

Sample Submission

Submit 3 ug of DNA, as measured by Qubit, diluted in 130 ul 10 mM Tris, pH 8 (Qiagen EB buffer) in 1.5 ul low-bind microfuge tubes are requested.  If you are able, please double this amount (6 ug in 260ul).  RNase I treatment of your DNA is highly recommended.

Quality Control

  1. 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.  
  2. Measure genomic DNA on the Nanodrop to evaluate purity. For a sample to pass QC 260/280 ~1.8, and 260/230 above 2.0 (or at least above 1.0). 
  3. Measure genomic DNA using fluorimetry to get an accurate sample concentration.  For a sample to pass QC, there must be ≥ 3ug (by fluorimetry)

Prior to sample submission please provide a gel image, fluorimetry results (ng/ul), and Nanodrop results (260/280, 260/230). If your samples don't pass QC please contact to us. It might still be possible to sequence them and we might have suggestions for you.

Library Creation

Steps include DNA shearing using the Covaris ultrasonicator, fragment purification, end polishing, and ligation to adaptors to generate libraries. The libraries are then normalized, and library molecules that represent coding regions are captured through hybridization to a probe. The captured libraries are then amplified using indexed (barcoded) primers. The size distribution of the final libraries is validated using the Agilent Bioanalyzer, the libraries are pooled equally, and the quantification of the pool is done using the qPCR-based KAPA Assay.

Sequencing

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


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.