This Genomics Services Laboratory (GSL) has over a decade of experience with next-generation sequencing (NGS) data generation and analysis. The GSL can assist investigators with multiple aspects of next-generation sequencing, including experimental design, library preparation, sequence generation, and data analysis. Advanced bioinformatics support for analyzing complex sequencing datasets, including integrated DNA and RNA sequence data, is a demonstrated strength.
NOVASEQ 6000 (v1.5 chemistry)
The NovaSeq 6000 system is Illumina’s latest advancement in NGS sequencing instrumentation, which combines v1.5 SBS chemistry with high throughput patterned flow cell technology to drastically reduce run times, simplify workflows and provide greater flexibility and increased throughput. The NovaSeq is a dual flow cell instrument with four patterned flow cell configurations (SP, S1, S2 and S4) providing 0.08 Tb – 6 Tb of total sequence data output. The XP chemistry allows for independent lane segregation. Each patterned flow cell contains billions of nanowells, allowing optimal high-density library cluster formation with increasing data output (Gb) per flow cell.
The Genomics Service Laboratory (GSL) is equipped with two NovaSeq 6000 instruments and operates with all flow cell configurations of paired-end 150 base-pair sequencing and XP lane segregation. This scalable operation allows the GSL to meet the demands of diverse sequencing projects from Genome Sequencing (GS) to RNA sequencing (RNA-seq).
Sequencing Output per Flow Cell
|NovaSeq 6000 System
|Flow Cell Type||SP||S1||S2||S4|
|2 x 50 bp||65-80 Gb||134-167 Gb||333-417 Gb||N/A †|
|2 x 100 bp||134-167 Gb||266-333 Gb||667-833 Gb||1600-2000 Gb|
|2 x 150 bp||200-250 Gb||400-500 Gb||1000-1250 Gb||2400-3000 Gb|
|2 x 250 bp||325-400 Gb||N/A †||N/A †||N/A †|
|Clusters Passing Filter|
|Single Reads||650-800 million||1.3-1.6 billion||3.3-4.1 billion||8-10 billion|
|Paired-end Reads||1.3-1.6 billion||2.6-3.2 billion||6.6-8.2 billion||16-20 billion|
Specifications based on Illumina PhiX control library. Yield and performance may vary depending on library type, sample quality, cluster density, and other experimental factors.
† NA: not applicable.
MISEQ (v3 chemistry)
Illumina MiSeq generates up to 15 Gb of 2 x 300 paired-end reads in < 3 days. The MiSeq sequencer is suited for targeted gene sequencing, small genome sequencing, metagenomics, and amplicon sequencing.
Sequencing Output per Standard Runs
|Read Length||Output||Reads Passing Filter|
|1 x 150 bp||3.3-3.8 Gb||22-25 million|
|2 x 75 bp||3.3-3.8 Gb||44-50 million|
|2 x 300 bp||13.2-15 Gb||44-50 million|
Illumina MiniSeq is well suited for small genomes, metagenomics, amplicon, and plasmid sequencing. The MiniSeq offers the flexibility to meet dynamic project needs with variable read lengths and data output options.
Sequencing Output per Standard Runs
|MiniSeq System High-Output Kit||MiniSeq System Mid-Output Kit|
|Read Length||2 x 150 bp||2 x 75 bp||2 x 150 bp|
|Output||6.6-7.5 Gb||3.3-3.75 Gb||2.1-2.4 Gb|
|Reads Passing Filter||44-50 million||14-16 million|
*Available upon request.
DNA Sequencing Services
All DNA samples are evaluated for concentration by Qubit® and quality on Agilent 4200 TapeStation.
Genome Sequencing (GS)
We provide genome sequencing for interrogating single-nucleotide variants (SNVs), insertions and deletions (indels), structural variants (SVs), and copy number variants (CNVs) in genomic coding and non-coding regions. Service includes DNA QC, library prep, and sequencing on Illumina platforms using paired-end 150 base-pairs reads.
- Human WGS sequence to yield an average 30X haploid coverage
- Human WGS sequence to yield an average 60X haploid coverage
- Custom options are available (including non-human samples). Please inquire.
Exome Sequencing (ES)
Exome sequencing targets the protein-coding genes (exons) in a genome. The human exome represents less than 2 % of the genome but contains ~85% of known disease-related variants. Enriching for exonic loci reduces the amount of required sequence data and provides a cost-effective approach to identifying disease-associated genomic alterations. Exome sequencing is a solution-based hybridization enrichment method using 5’biotin-modified oligonucleotide probes to capture genomic regions of interest (exons). Service includes genomic DNA QC assessment, exome capture of DNA library, and sequencing on Illumina platforms as paired-end reads.
- Human WES sequence to yield an average 100-150X depth of coverage
- Human WES sequence to yield an average 250X depth of coverage
- Mouse exome capture is available.
RNA Sequencing (RNA-Seq) Services
All RNA samples are evaluated for concentration by Qubit® and integrity using Agilent 2100 Bioanalyzer, or the Perkin Elmer LabGX.
Total RNA-Seq with Ribosomal Reduction or Poly-A Selection
RNA-Seq measures gene and transcript abundance and identifies known and novel features of the transcriptome. This service is for differential expression of RNA from Human/Rat/Mouse and other non-human organisms. Total RNA-seq with ribosomal RNA reduction removes rRNA from total RNA samples. In contrast to poly(A) selection enrichment, the rRNA reduction approach allows for the preservation of non-polyadenylated RNAs, including immature mRNAs and ncRNAs. Poly(A) selection is available on request. This service includes total RNA QC assessment, ribosomal RNA depletion or poly(A) selection, library construction with the addition of an external ERCC spike-in control (https://www.thermofisher.com/order/catalog/product), and sequenced on Illumina platform to generate 60-80 million150bp paired-end reads for human/mouse/rat samples. RNA-seq is also available for non-human/mouse/rat samples.
Small non-coding RNAs play a role in gene silencing and post-transcriptional regulation of gene expression. Small/miRNA sequencing provides the ability to measure and compare the expression profiles of known miRNAs and other small non-coding RNAs. This service includes initial RNA QC assessment, library construction, size selection by Pippin Prep instrument, and sequencing on Illumina MiSeq or MiniSeq to generate 4 million 75bp paired-end reads per sample.
How to Submit Samples
1. Initiate and Submit your request via iLab.
- Login or Register for an iLab account, https://nch.corefacilities.org/landing/19
- Under Core Facilities, select Nationwide Children’s Hospital (NCH) and Genomics Services Laboratory (GSL)
- Initiate the Next Generation Sequencing Request
- Enter the project description information on NGS Form
- The GSL will issue a quote upon completion of your request
- Agree to quote and provide payment information
- You must have payment information supplied before work on samples will begin
2. The core will assess nucleic acid integrity and concentration and may request replacements of sub-optimal samples. See Sample Submission Guidelines section on specifics of how to submit samples.
3. Ship or deliver your samples. If delivering your samples in person, please contact IGM Genomic Services to schedule a specific day and time. The Genomic Services Laboratory location is in Research Building 3, WB2160, on NCH main campus.
575 Children’s Crossroads, WB2160
The Steve and Cindy Rasmussen Institute for Genomic Medicine
Abigail Wexner Research Institute at Nationwide Children’s Hospital
Columbus, Ohio 43215
*Please contact IGMGenomicServices@NationwideChildrens.org for questions regarding service offerings and delivery of samples.
Hours of Operation
Regular hours are 8:00 a.m. to 4:30 p.m., Monday through Friday.
Sample Submission Guidelines
Please submit samples in Eppendorf 1.5 ml LoBind tubes (Fisher Sci; Cat#13-698-791), or Eppendorf™ twin.tec™ 96 Well LoBind PCR Plates, Skirted (Fisher Sci; Cat# E0030129512). Tubes should be clearly labeled and readable with GSL order number, sample identifier, and lab name/PI.
For RNA/DNA QC services, submit samples in 0.5ml PCR strip tubes.
Samples may be suspended in dH2O, 10mM Tris, or Qiagen EB. High salt (EDTA) concentration can interfere with the various enzymes that are used during library preparation. Our preference is that samples are suspended in dH2O.
Library Preparation Services
|Service||Library Prep Method||Input Material||Concentration||Minimum volume required||Recommended amount|
|Human Whole Exome Sequencing (WES)||NEBNext Ultrall FS Library Prep Captured using IDT xGEN Exome Research Panel v2 + Copy Number Variant Probes||genomic DNA||>25 ng/µL||20 µL||≥200 ng|
|Mouse Whole Exome Sequencing (WES)||Agillent SureSelect QXT with Mouse ALL Exon Capture||genomic DNA||>20 ng/µL||20 µL||≥200 ng|
|Whole Genome Sequencing (WGS)||NEBNext Ultrall FS||genomic DNA||>25 ng/µL||20 µL||≥500 ng|
|FFPE DNA - WGS/WES||NEBNext Ultrall FS||genomic DNA||>25 ng/µL||30 µL||≥1000 ng|
|Adaptive immunSEQ (human)||hsTCRB kit||genomic DNA||10-90 ng/µL||50-100 µL||Dependent on Sample Type|
|FFPE RNA - Total Stranded RNA-seq||NEBNext Ultrall II Directional||Total RNA||>50 ng/µL||20 µL||≥1000 ng|
|Stranded Total RNA-Seq||NEBNext Ultrall II Directional||Total RNA||>50 ng/µL||20 µL||≥500 ng|
|miRNA-Seq||NEBNext Small RNA||Total RNA including small RNA fractions (>17NT)||>50 ng/µL||25 µL||≥1000 ng|
Sample Submission Guidelines for Sequencing Services (client-submitted libraries)
|Illumina Sequencer||Sample Type||Minimum Volume||Minimum Concentration|
|MiniSeq||Final Library||20 µl||1 nM|
|MiSeq||Final Library||20 µl||10 nM|
|HiSeq 4000||Final Library||20 µl||3 nM|
|NovaSeq 6000: SP-Standard||Final Library||220 µl||2.25 nM|
|NovaSeq 6000: SP-XP (2-lane segregation)||Final Library||20 µl per lane||1.5 nM|
|NovaSeq 6000: S1-STandard||Final Library||220 µl||2.25 nM|
|NovaSeq 6000: S1-XP (2-lane segregation)||Final Library||20 µl per lane||1.5 nM|
|NovaSeq 6000: S2-Standard||Final Library||320 µl||2.25 nM|
|NovaSeq 6000: S2-XP (2-lane segregation)||Final Library||25 µl per lane||1.5 nM|
|NovaSeq 6000: S4-Standard||Final Library||620 µl||2.25 nM|
|NovaSeq 6000: S4-XP (4-land segregation)||Final Library||35 µl per lane||1.5 nM|
The following items to be completed prior to dropping off samples:
- iLab request completed with payment
- Sample Submission Form and Project Intake Form will be provided by the Genomic Services Laboratory via email. Please fill out and email IGM Genomic Services alias and/or upload to the iLab order
- Please label all sample tubes and forms with iLab GSL order number
The IGM Computational Genomics Group has partnered with the GSL to provide the substantial technical and bioinformatics expertise required to oversee the multiple platforms that acquire, store and analyze large and complex genomic data sets. The Translational Bioinformatics Team provides bioinformatics services on a collaborative basis at subsidized hourly rates and serves as an interface between the research investigator and the multiple domains that are required to handle the size and complexity of genomic data. IGM has built a dedicated, cloud-based computational resource through Amazon Web Services (AWS), allowing large HPC or Hadoop clusters with hundreds of instances to be provisioned on demand, enabling us to support the analysis needs of both large and small scale sequencing projects. We are uniquely positioned to adapt and develop new computational tools in a manner that is driven by the demands of our research community as it utilizes new technologies.
The Computational Genomics Group has developed state of the art analytical pipelines for NGS analysis including, but not limited to, transcriptomics (RNA-Seq and miRNA-Seq), whole genome sequencing (WGS), whole exome sequencing (WES) and paired tumor-normal somatic variant detection. Through development of the balanced parallelization strategy for human genome analysis (named “Churchill”), we were successfully able to reduce the time taken to go from raw sequencer output to final variant calls from days to under two hours, enabling both rapid and accurate identification of disease causing genetic variants in germline and cancer samples. In addition to developing our own bioinformatics tools, the unit also evaluates and supports multiple software products for genomics applications that are designed to make data analysis tools accessible to biologists.
Our facility does not expect authorship on studies using data generated on a fee-for-service basis. However, fee-for-service reimbursement for bioinformatics support does not alter the contribution to the academic enterprise, and co-authorship should be considered. Fee-for-service bioinformatics support is simply an alternative to salary support as would be provided by serving as a co-investigator on an NIH grant, for example. Consideration for authorship should be based on the accepted criteria of most medical journals with the contribution of each person being evaluated as a manuscript is prepared. These criteria generally cite both study design and bioinformatics analysis as intellectual input sufficient for authorship. It is impossible to define every situation in advance; however, it should be clear that reimbursement for time does not preclude or replace authorship.
IGM Publications resulting from data generated in IGM Genomic Services Core should cite the Nationwide Foundation Pediatric Innovation Fund as well as the The Ohio State University Comprehensive Cancer Center – Arthur G. James Cancer Hospital and Richard J. Solove Research Institute (OSUCCC – James) P30 grant P30 CA016058 which provides funding for our core.
Methods: “[Service] was performed by the IGM Genomic Services Lab of The Research Institute at Nationwide Children’s Hospital, Columbus, Ohio”
Acknowledgements: “We thank [name(s) of personnel] of the IGM Genomic Services Lab of the Research Institute at Nationwide Children’s Hospital, Columbus, Ohio for their help with [help provided]”