Library Preparation

RNA & DNA Library Preparation

The Genomics Core offers full library prep services for many sample types and applications for the Illumina HiSeq and Miseq platforms. If the library prep that is of interest to you is not listed below, please contact the Core Manager, Simone Edelheit to discuss your project.

Sample Submission Requirements 

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For specifics on total amount and volume required, please see RNA and DNA library preparation options in the charts below Submit all samples in 1.5ml eppendorf tubes, clearly labeled ON TOP of tube with sample name and user name/initials. Label the SIDE of tube with the date. Sample name on tube and iLab request form should match. Large volume projects may be submitted in FULL SKIRT 96-well plates, with at least one SIDE WALL clearly labeled with project name, plate number, user name, and date. Sample names and well location will be submitted through iLab. 


RNA Library Preparation Options

All RNA submitted to the Core for library processing will undergo quality control (QC) to make sure it is of high enough quality and quantity to move forward with library preparation. The QC of each sample includes Qubit for concentration (ng/ul) and the Agilent Bioanalyzer RNA chip to determine the RNA Integrity Number (RIN). The cost of the QC is included in the price of library prep. If a sample fails QC, the user will be charged for QC portion only. The Core will provide expert advice as to whether or not an RNA sample should be used for downstream analysis, but the final decision will be made by the user. Please see the NGS Run Guarantees Section for additional information. 

Since 2015, the Core has partnered with the Applied Functional Genomics Core (Director: Dr. Mark Cameron) from the Department of Population and Quantitative Health Sciences, to provide RNAseq analysis from ultra-low RNA input amounts. Contact the Core for more details. 

For a list of available preps and pricing, view our Pricing page.

RNA Library Prep Kit Description Amount Strongly Recommended Amount Required (per Qubit) Typical Run Type & Coverage
Illumina TruSeq Stranded Total RNA w/ Ribo Zero Gold
  • Whole-transcriptome analysis
  • Generates directional RNA-focused libraries
  • Low-quality and FFPE compatible
  • Removes both cytoplasmic and mitochondrial rRNA
  • Up to 96 unique indexes
  • Use with standard input RNAs (>15ng/ul)
  • 50ng/ul in ≥20ul
  • RIN: 7.0 - 10.0 

     

  • 15ng/ul in 20ul
  • RIN: 2.0-10.0
  • NextSeq 550 1x75bp for Gene Expression only (25-50M reads per sample)
  • NextSeq 550 2x150bp for Alternative Splicing (>50M reads per sample)
Low and Ultra-low RNA library Prep
  • The Core and user will work directly with the Applied Functional Genomics Core.
  • TBD
  • TBD
  • TBD

Submit a Request


DNA Library Preparation Options

All DNA submitted to the Core for processing will undergo quality control (QC) to make sure it is of high enough quality and quantity to move forward with library preparation. The QC of each sample includes Qubit for concentration (ng/ul) and agarose gel electrophoresis to check quality. The cost of the QC is included in the price of library prep. If a sample fails QC, the user will be charged for QC portion only. The Core will provide expert advice as to whether or not a DNA sample should be used for downstream analysis, but the final decision will be made by the user. Please see the NGS Run Guarantees Section for additional information. 

For a list of available preps and pricing, view our Pricing page.

DNA Library Prep Kit Description Requirements   (per Qubit) Typical Run Type & Coverage
Illumina TruSeq DNA PCR Free
  • WGS of small genomes to whole-human genome sequencing
  • Shortened GEL-FREE workflow for reduced library bias and gaps
  • Up to 96 unique indexes
  • 100ng/ul in 20ul
  • 260/280: 1.8-2.0
  • NextSeq 2x150bp
  • MiSeq v3 2x250bp
Nextera XT DNA
  • Prepare libraries for small genomes, PCR amplicons, and plasmids to sequence using MiSeq technology Multiplex up to 384 samples
  • 10ng/ul in 20ul
  • 260/280: 1.8-2.0
  • NextSeq 2x150bp
  • MiSeq v3 2x250bp
Nextera Rapid Capture Exome
  • All-in-one kit for library prep and exome enrichment
  • Includes 37Mb of expertly selected exonic- content
  • Up to 24 unique indexes
  • 10ng/ul in 20ul
  • 260/280: 1.8-2.0
  • NextSeq 2x150bp
  • 100X coverage = 80M Paired reads per sample
TruSeq Exome / TruSeq Rapid Exome
  • All-in-one kit for library prep and exome enrichment
  • Includes 45Mb of exonic content
  • Up to 12 unique indexes 
  • TruSeq Exome is FFPE compatible. 
  • 10ng/ul in 20ul
  • 260/280: 1.8-2.0
  • NextSeq 2x75bp
2-Step PCR Amplicon Approach (CRISPR)
  • Best used for CRISPR/Cas gene editing validation
  • Tailed amplicon sequencing using Nextera Dual indexing adapters and the MiSeq platform
  • Up to 288 unique indexes available. 
  • 10ul PCR product
  • One PCR per well
  • Gel Image
  • MiSeq v3 2x250bp
16S Metagenomics (rRNA Sequencing)
  • Amplicon sequencing method used to identify and compare bacteria present within a given sample
  • Primer pair sequences for the V3 and V4 region that create a single amplicon of approximately ~460 bp
  • Up to 384 unique indexes. 

Either:

  • DNA – 10ng/ul in 20ul
  • V3/V4 PCR product – 25ul w/ gel image
  • MiSeq v3 2x250bp
  • ~100,000 reads per sample
Illumina TruSeq Custom Amplicon Kit
  • Fully customizable, amplicon-based assay for targeted resequencing
  • Multiplex up to 1536 amplicons per reaction to cover up to 650 kb of cumulative sequence
  • Pool up to 96 samples for maximum benefit from the MiSeq v3 reagents
  • Access automated, design-specific variant calling and final data analysis using the Illumina MiSeq software
  • FFPE DNA compatible
  • 10ng/ul in 20ul
  • 260/280: 1.8-2.0
  • Illumina QC Kit required for FFPE samples
  • MiSeq v3 2x250bp
  • Custom analysis workflow provided for MiSeq run.
  • Work with Illumina Sales Representative for ordering custom project.

 

Submit a Request