GENOME TECHNOLOGY ACCESS CENTER • DEPARTMENT OF GENETICS • WASHINGTON UNIVERSITY SCHOOL OF MEDICINE

Next Generation Sequencing (NGS)

How to Get Started

To obtain a quote or discuss a project, contact us via our project initiation form.

If you would like to submit a sample, please review our sample submission guidelines at the bottom of this page and contact us for submission instructions.  Below you will find more information about our sequencing services: library preparation and sequencing.

Library Preparation

GTAC offers a wide selection of library prep types for Illumina sequencing.  Below is a list of prep types that we standardly offer.  For further description of these prep types and/or a consultation with our team to discuss experimental design, please contact genetics-gtac@email.wustl.edu

  • Whole Genome Sequencing
  • Whole Exome/targeted Sequencing
  • mRNA-Seq
  • Small RNA-Seq
  • Single-cell RNA-Seq
  • ChIP-Seq
  • Amplicon-Seq
  • Bacterial 16S-Seq

For CAP/CLIA accredited services please visit Genomics and Pathology Services at http://wugps.wustl.edu/


Sequencing

GTAC operates Illumina NovaSeq 6000, HiSeq 3000, HiSeq 2500, and MiSeq sequencers. The NovaSeq is the newest instrument capable of generating billions of reads overnight. The NovaSeq 6000 and HiSeq 2500 can run two flowcells simultaneously, and the HiSeq 3000 runs one flowcell at a time.  DNA fragments can be read from one or two ends (paired-end).

For the HiSeq 2500, we currently offer 2×101 paired-end and 1×50 single read sequencing runs. Both run types are standardly offered in rapid mode, where each flowcell has two lanes, and each lane averages 150 million reads or more.  Other run types can be accommodated if you can fill at least two lanes at a time; that is, fill a flow cell.

For the HiSeq 3000, we offer 1×50 single read and 2×150 paired-end sequencing runs.  Each flowcell has 8 lanes, and each lane averages 350 million reads for standard high quality, high diversity libraries.  Other run types can be accomodated if you can fill all eight lanes at a time; that is, fill a flow cell.

For NovaSeq, you can choose the flowcell to tailor to the number of reads you need, and pair that with the number of cycles for your read length. The S1 and S2 flowcells come with either 100, 200 or 300 cycles and the S4 is available with 200 or 300 cycles.

GTAC also operates Illumina MiSeq instruments. The MiSeq runs one single-lane flow-cell per run. Each run is capable of producing 12-15 million reads. We currently offer 2×150 paired end and 2×250 paired end sequencing runs in version 2 chemistry.

 
More information about the sequencing instruments can be found here: NovaSeq HiSeq 2500 HiSeq 3000 Illumina-MiSeq

Sample Submissions Guidelines

Below are guidelines for sample submission and descriptions of the library preparation services. Please state if any of these requirements are not met before submitting. If interested in fragment size other than 150-300bp please specify.  If interested in other library preparations please contact us at genetics-gtac@email.wustl.edu

genomic DNA for Genome, Exome or Targeted Capture Sequencing

Guidelines for Submission:

  • Buffer = TE
  • Quantity = 1-3ug
  • 260/280 = >1.7
  • 260/230 = >2
  • If available please submit an Agarose Gel picture to verify DNA quality

Description of Service:

Whole Genome Sequencing

Whole genome sequencing determines the complete DNA sequence of an organism’s genome.  Whole genome sequencing using NGS technologies provides information on a genome that is orders of magnitude larger than that provided by the current leaders in genotyping technology.

Whole Exome Sequencing

Agilent SureSelect Exome
The SureSelect Human Exome kits are is designed to target all human exons, regions totaling approximately 50-54Mb, all in a single tube. This prep covers 1.22% of human genomic regions corresponding to the CCDS exons. We currently offer Agilent’s version 5 and Clinical Research Exome – please inquire if interested in a different design. We also offer Agilent’s Mouse All Exon kit that is 50Mb.

Custom gDNA Capture

Agilent SureSelct Target Enrichment
The SureSelect Target Enrichment kit allows the flexibility to design a target from < 200kB to + 34Mb using Agilent’s eArray tool or their design service. This method allows you to focus on just the regions you are interested in.

Total RNA for RNA-seq

Guidelines for Submission:
•   Buffer = DEPC water
•   Quantity = 5ug-10ug (polyA selection), 1ug-5ug (Ribo-depletion), 1ug (Small RNA), 10-30ng (low input Takara -Clontech SMARTer or Sigma Seqplex).  Lower input amounts are possible – contact us for options for lower input or degraded samples.)
•   260/280 at least 1.9
•   RIN value at least 8.0
•   If available please submit Agilent RNA chip electropherogram

Description of Service:

Polyadenylation
mRNA is extracted form total RNA using a Dynal mRNA Direct kit.  The Oligo-dT beads pull the mRNA out of solution by binding with the poly (A) tail. mRNA is then fragmented and reverse transcribed to double stranded cDNA with random primers before addition of adapters. This method requires a higher input amount of intact total RNA and only works for species that have polyadenylation.

Depletion
RNA depletion using Ribo-Zero is highly effective in the removal of rRNA with either high-quality or partially degraded RNA samples starting with less input RNA than Poly (A) selection.  Removes 28S, 18S, 5.8S, and 5S rRNA from solution. This method preserves the transcript profile of the sample, and is capable of recovering both 5’ and 3’ ended fragments in partially degraded samples. This kit has only been tested on a subset of species (Species Compatibility).

cDNA Amplification
Using Clontech’s SMARTer kits allows the preparation of cDNA libraries from total RNA for sequencing.  A library can be created with picogram amounts of total RNA. The kit uses poly (A) based priming to amplify cDNA from total RNA, therefore it requires intact starting material.
Using Sigma’s Seqplex kit allows for preparation of cDNA libraries from picogram amounts of total RNA. This method uses “quasi-random” priming and can be used on RNA that is degraded.

Single Cell RNAseq (FACSseq)
This method generates cell-barcoded cDNA from single sorted cells that is then pooled and amplified. A sequencing library is generated from the 3′ fragments that contain the barcode. This technique uses an oligo-dT priming approach, therefore, this method is only compatible with polyadenylated species. The data generated will be at the gene level, not whole transcriptome due to the positioning of the 3′ barcode and the short read technology. Please contact us for details and for requirements for submission.

Single Cell RNAseq (10X Chromium)
This method generates cell-barcoded cDNA from single cells captured into droplets utilizing the 10X Chromium system. A sequencing library is generated from the 3′ fragments that contain the barcode. This technique uses an oligo-dT priming approach, therefore, this method is only compatible with polyadenylated species. The data generated will be at the gene level, not whole transcriptome due to the positioning of the 3′ barcode and the short read technology. Please contact us for details and for requirements for submission.

Small RNA
Sequencing of microRNAs requires a separate library prep that preserves the very short species as other preps include purification steps where this material can be lost. This library prep uses adapter ligation onto the RNA before cDNA is prepared. The small RNA library fragments are selected from the remaining total RNA by a size selection targeting the 22-30 nucleotide micro RNAs.

ChIP DNA

Guidelines for Submission:
·       Buffer = H2O
·       Quantity = 10-20ng within the 150-500bp size range
·       Highly recommend sending an Agarose Gel picture or bioanalyzer electropherogram to verify DNA size 

Description of Service:

ChIP-seq combines chromatin immunoprecipitation(ChIP) with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins.  As little as 10ng of ChIP-DNA fragments are submitted and prepped for Illumina sequencing. We have also used this prep to create libraries from Formaldehyde-Assisted Isolation of Regulatory Elements samples (FAIRE-Seq).

Amplicon-Seq

Guidelines for Submission:
·       Buffer = TE or Water
·       Quantity at least 100ng
·       Concatemerized Equimolar pool of Amplicons from each sample

Description of Service:

Amplicon sequencing consists of sequencing known regions of interest.  This highly targeted approach enables a wide range of applications for discovering, validating, and screening genetic variants in a rapid and efficient manner. We can accommodate amplicons spanning exons as we request the amplicons be concatemerized, and we randomly fragment the ligated product.

16S-Seq

Guidelines for Submission:
·       Buffer = TE or Water
·       Quantity 25-100ng
·       Recommend sending an Agarose Gel picture or Bioanalyzer electropherogram to verify DNA size.

Description of Service:

GTAC will employ the high-throughput capability of Fluidigm Dynamic Array integrated fluidic circuits, allowing for sequence library generation for up to 48 or 192 samples in a single run.  Ready-to-sequence amplicons can be generated for one or up to all nine ribosomal hyper-variable regions of bacterial 16S ribosomal RNA.  Multiple hyper-variable region analysis is executed with the MVRSION tool.  Analysis at the single region can be done using QIIME (pronounced “chime” –  Quantitative Insights Into Microbial Ecology).