Next Gen Sequencing

DNA Sequencing is the process of determining the order of the four nucleotide bases adenine (A), thymine (T), cytosine (C), guanine (G) that made up the DNA molecules. Sequencing allows scientists to determine the whole organism’s genetic information and also what information is being carried in a particular DNA segment. With the unprecedented access to genomic information, NGS made it possible for expediting breakthroughs in human healthcare, discovering complex disorders and genetic variation underlying cancers, identifying pathogenic and susceptibility genes, analysing gene diversity and evolution. 

VANTAGE NGS services are executed with the latest technologies and bioinformatics enable us to deliver high-quality data at rapid turnaround time. We offer a variety of services ranging from Whole Genome Sequencing, Whole Exome Sequencing, Metagenomics, and Epigenomic Sequencing. All sequencing will be performed using Illumina platforms, NovaSeq XP Plus, NovaSeq 6000 and  MiSeq targeting different amounts of reads/samples.

• Human/Mouse/Bacteria
  • Whole Genome Sequencing
    • High-pass WGS
    • Low-pass WGS
  • Whole Exome Sequencing
    • TruSeq/IDT WES
    • Twist WES
• Epigenetic
  • Whole Genome Bisulfite Sequencing (Human and Mouse)
  • ATAC-Sequencing
• Metagenomic 
  • 16s Amplicon V4
  • Shotgun Metagenomic WGS
• Immunology
  • TruSight Myeloid Sequencing
• DNA-Protein Interaction
  • ChIP-Seq library prep and sequencing

All samples are performed by highly trained and experienced laboratory professionals. We follow strict quality procedures to ensure your samples have the best result as possible. Below is this description of a regular sequencing workflow.

DNA Sequencing Workflow

1. Creating Project 
   • Create Project using iLab https://vanderbilt.corefacilities.org/
     • For non-Vanderbilt customers, please sign-up as non-Vanderbilt user

•  
  •  
    • Once sign-in, search for VUMC VANTAGE to request service
    • For detail instruction please view here: pdf
  • VANTAGE Biobanking Lab also offers nucleic DNA and RNA extractions from samples such as whole blood, Buccal swabs, Saliva samples, cells, various tissues as well as Formalin-Fixed Paraffin-Embedded (FFPE) samples. Check here for more information

2. Sample Requirement

All samples received for NGS will be QC using fluorometry Qubit or Picogreen and integrity check using BioAnalyzer or TapeStation. All QC results will be posted for review and approval.  If the quantity and quality fall within the protocol specifications, VANTAGE will continue with library preparation.

3. Library Preparation 

4. QC Check and Balancing

Library Quality Control analysis will be performed by using Qubit and BioAnalyzer to determine the concentration and size bp. Each library that passes the final QC undergoes qPCR using the KAPA library quantification kit and QuantStudio. qPCR allows highly accurate quantification of library molecules that can seed clusters and balance target sample representation during pooling. Multiplexed samples are pooled in equimolar or user-defined ratios to achieve the multiplexing level requested by the user using Illumina MiSeq platform.

5. Sequencing

Final pooling and rebalancing to ensure target sample representation and user defined ratio are achieved. Sequencing will be performed at Paired-End 150 bp on the Illumina NovaSeq 6000 targeting different reads per sample base on type of assays

6. Data Analysis and Delivery 

QC reports for the sequencing quality and the per sample yield will be provided.  The file deliverable is a demultiplexed FASTQ containing the PF reads. Intermediate analysis is also available through Illumina’s Dragen RNASeq pipeline.

• Whole Genome Sequencing (Human or Mouse)

A Quality Control analysis will be performed on a genomic DNA sample and the results will be posted for review and approval. If the quantity and quality fall within the protocol specifications, uniquely dual indexed libraries will be prepared. Whole genome sequencing will be performed at PE 150 on the NovaSeq 6000, targeting an average of 30X coverage per sample. QC reports for the sequencing quality and the per sample yield will be provided.  The file deliverable is a demultiplexed FASTQ containing the PF reads. Further bioinformatics services are available upon request.

• Whole Genome Bisulfite (Human or Mouse)

A Quality Control analysis will be performed on the whole genomic DNA sample and the results will be posted for review and approval. If the quantity and quality fall within the protocol specifications, uniquely dual indexed WGBS libraries will be prepared. Whole genome bisulfite sequencing will be performed at PE 150 on the NovaSeq 6000, targeting an average of 40X coverage per sample. QC reports for the sequencing quality and the per sample yield will be provided.  The file deliverable is a demultiplexed FASTQ containing the PF reads. Further bioinformatics services are available upon request.

• HP-WGS/LP-WGS:

A Quality Control analysis will be performed on the whole genomic DNA sample and the results will be posted for review and approval. If the quantity and quality fall within the protocol specifications, uniquely dual indexed libraries will be prepared. One aliquot of the library will be used for WES capture.  Whole genome sequencing will be performed at PE150 on the NovaSeq 6000, targeting an average of 1-2X coverage per sample. Sequencing will be performed at PE150 bp on the Illumina NovaSeq 6000, targeting an average of 20M reads/sample (50X coverage) for the captured samples. QC reports for the sequencing quality and the per sample yield will be provided.  The file deliverable is a demultiplexed FASTQ containing the PF reads. Further bioinformatics services are available upon request.

• Truseq/IDT Whole Exome Sequencing

• Standard 100x (40M reads/sample)

• Standard 50X (20M reads/sample)

A Quality Control analysis will be performed on the whole genomic DNA sample and the results will be posted for review and approval. Library preparation and capture will be performed utilizing the XGen Research Panel probes from IDT.  Sequencing will be performed at Paired-End 150 bp on the Illumina NovaSeq 6000, targeting an average of 40M reads/sample (100X coverage). QC reports for the sequencing quality and the per sample yield will be provided.  The file deliverable is a demultiplexed FASTQ containing the PF reads. Further bioinformatics services are available upon request.

• Twist Whole Exome Sequencing

• Standard 100x (40M reads/sample)

• Standard 50X (20M reads/sample)

A Quality Control analysis will be performed on the whole genomic DNA sample and the results will be posted for review and approval. Library preparation and capture will be performed utilizing the Twist Exome library prep and capture kit.  Sequencing will be performed at Paired-End 150 bp on the Illumina NovaSeq 6000, targeting an average of 20M reads/sample (50X coverage). QC reports for the sequencing quality and the per sample yield will be provided. The file deliverable is a demultiplexed FASTQ containing the PF reads along with the following files generated using the Edico Dragen Enrichment pipeline: Aligned reads - BAM format; Small variant calls - VCF, gVCF; Optional: Structural Variants - VCF; Optional: CNV - GFF3, VCF.  Interpretation of the analysis results is also available upon request.          

• Shotgun Metagenomics WGS:

A Quality Control analysis will be performed on the genomic DNA sample and the results will be posted for review and approval. If the quantity and quality fall within the protocol specifications, uniquely dual indexed libraries will be prepared using the Nextera DNA Flex kit. Whole genome sequencing will be performed at PE150 on the NovaSeq 6000, targeting an average of 500,000-1M reads per sample. QC reports for the sequencing quality and the per sample yield will be provided.  The file deliverable is a demultiplexed FASTQ containing the PF reads. Further bioinformatics services are available upon request.

• 16S Amplicon:

The DNA will be quantified and used for library construction. Metagenomic libraries will be generated using primers targeting the V4 region of the 16S rRNA gene.  Sequencing will be performed on the Illumina MiSeq V2 at Paired-End 250bp. The file deliverable is a demultiplexed FASTQ containing the PF reads. 

• ChIP-Seq Library preparation and sequencing 

A Quality Control analysis consisting of quantitation only will be performed on the ChIP DNA and the results will be posted for review and approval. If the quantity is within the protocol specifications, uniquely dual indexed libraries will be prepared. ChIP Seq sequencing will be performed at PE 150 on the NovaSeq 6000, targeting an average of 50M reads per sample. QC reports for the sequencing quality and the per sample yield will be provided.  The file deliverable is a demultiplexed FASTQ containing the PF reads. Further bioinformatics services are available upon request.

• TruSight Myeloid

A Quality Control analysis will be performed on the DNA sample and the results will be posted for review and approval. If the quantity and quality fall within the protocol specifications, VANTAGE will perform library preparation utilizing the TruSight Myeloid Sequencing Panel. Sequencing will be performed at Paired-End 150 bp on the Illumina NovaSeq 6000 targeting an average of 1M reads per sample. Data will be analyzed using the TruSeq Custom Amplicon BaseSpace App for automated alignment and somatic variant calling. The deliverable files include: a demultiplexed FASTQ containing the PF reads, a Bam file containing the aligned reads and a vcf containing the called variants. Further bioinformatics services are available upon request.

RNA Sequencing is a powerful methodology for studying the transcriptome qualitatively and quantitatively. It can develop transcriptional profiling, SNP identification, and differential gene expression in biological samples under specific conditions. Understanding the transcriptome is key to identify protein expression in a cell and evaluate changes that may indicate potential pathogenic development.

VANTAGE offers a variety of RNA-Seq transcriptome services. All samples are sequenced and performed at multiplex Paired-End 150 bp on the Illumina NovaSeq 6000. Customers are provided with statistical reports for sequencing quality and per sample yield. The file deliverable is a demultiplexed FASTQ containing the PF reads. Intermediate analysis is also available through Illumina’s Dragen RNA Seq pipeline. Our processing and support provide excellent genomics technology and service to the Vanderbilt community and many other investigators.

• Transcriptome sequencing
  • Standard mRNA
  • Total RNA
  • Low input RNA (ovation)
  • miRNA (Small RNA)
• Immunology
  • Archer-TCR/BCR

All samples are performed by highly trained and experienced laboratory professionals. We follow strict quality procedures to ensure your samples have the best result as possible. Below is this description of a regular sequencing workflow.

RNA Sequencing Workflow

1. Creating Project
   • Create Project using iLab https://vanderbilt.corefacilities.org/
     • For non-Vanderbilt customers, please sign-up as non-Vanderbilt user
     • Once sign-in, search for VUMC VANTAGE to request service
     • For detail instruction please view here: pdf
   • VANTAGE BioBanking Lab also offers nucleic DNA and RNA extractions from samples such as whole blood, Buccal swabs, Saliva samples, cells, various tissues as well as Formalin-Fixed Paraffin-Embedded (FFPE) samples. Check here for more information

2. Sample Requirement

Sample Quality Control analysis will be performed by using fluorometry Qubit or Picogreen and integrity by BioAnalyzer or TapeStation. All QC results will be posted for review and approval.  If the quantity and quality fall within the protocol specifications, VANTAGE will continue with library preparation.

 

3. Library Preparation 

4. QC Check and Balancing

Library Quality Control analysis will be performed by using Qubit and BioAnalyzer to determine the concentration and size bp. Each library that passes the final QC undergoes qPCR using the KAPA library quantification kit and QuantStudio. qPCR allows highly accurate quantification of library molecules that can seed clusters and balance target sample representation during pooling. Multiplexed samples are pooled in equimolar or user-defined ratios to achieve the multiplexing level requested by the user using Illumina MiSeq platform.

5. Sequencing

Final pooling and rebalancing to ensure target sample representation and user defined ratio are achieved. Sequencing will be performed at Paired-End 150 bp on the Illumina NovaSeq 6000 targeting an average of 50M reads per sample. 

6. Data Analysis and Delivery

QC reports for the sequencing quality and the per sample yield will be provided.  The file deliverable is a demultiplexed FASTQ containing the PF reads. Intermediate analysis is also available through Illumina’s Dragen RNA Seq pipeline. 

• Standard mRNA 

A Quality Control analysis will be performed on the RNA sample and the results will be posted for review and approval.  If the quantity and quality fall within the protocol specifications, VANTAGE will perform mRNA enrichment and cDNA library preparation utilizing the stranded mRNA (polyA-selected) library preparation kit.  Sequencing will be performed at Paired-End 150 bp on the Illumina NovaSeq 6000 targeting an average of 50M reads per sample. QC reports for the sequencing quality and the per sample yield will be provided.  The file deliverable is a demultiplexed FASTQ containing the PF reads. Intermediate analysis is also available through Illumina’s Dragen RNA Seq pipeline. 

• Total RNA

A Quality Control analysis will be performed on the total RNA sample and the results will be posted for review and approval. If the quantity and quality fall within the protocol specifications, VANTAGE will perform library preparation utilizing a ribo-depletion total RNA library preparation kit.  Sequencing will be performed at Paired-End 150 bp on the Illumina NovaSeq 6000 targeting an average of 50M reads per sample. QC reports for the sequencing quality and the per sample yield will be provided.  The file deliverable is a demultiplexed FASTQ containing the PF reads. Intermediate analysis is also available through Illumina’s Dragen RNA Seq pipeline. 

• Low input RNA - 50M reads/sample

A Quality Control analysis will be performed on the total RNA sample and the results will be posted for review and approval.  If the quantity and quality fall within the protocol specifications, VANTAGE will perform RNA amplification using the Ovation RNA Seq System V2 kit and cDNA library preparation utilizing the NEB library preparation kit.  Sequencing will be performed at Paired-End 150 bp on the Illumina NovaSeq 6000 targeting an average of 50M reads per sample. QC reports for the sequencing quality and the per sample yield will be provided.  The file deliverable is a demultiplexed FASTQ containing the PF reads. Further bioinformatics services are available upon request. 

• miRNA (Small RNA):

A Quality Control analysis will be performed on the total RNA sample and the results will be posted for review and approval.   If the quantity and quality fall within the protocol specifications, VANTAGE will perform miRNA library preparation utilizing NextFlex sample prep kit. The samples will be sequenced on NovaSeq 6000 (targeting ~10M reads/sample). QC reports for the sequencing quality and the per sample yield will be provided.  The file deliverable is a demultiplexed FASTQ containing the PF reads. Further bioinformatics services are available upon request. 

• Archer-TCR/BCR:

A Quality Control analysis will be performed on the total RNA sample and the results will be posted for review and approval.   If the quantity and quality fall within the protocol specifications, VANTAGE will perform ArcherDX-TCR library prep using ArcherDX reagents. Sequencing will be performed at Paired-End 150 bp on the NovaSeq 6000, targeting an average of 4M reads per sample. QC reports for the sequencing quality and the per sample yield will be provided.  The file deliverable is a demultiplexed FASTQ containing the PF reads. Further bioinformatics services are available upon request.

The 10X Genomics Chromium Controller is a single-cell profiling technology that enables the analysis of large cell numbers at a high capture efficiency (of up to 65%). The platform allows for high-throughput single cell transcriptome, immunome, or copy number analysis in a variety of cell types as well as single-cell nuclei. The flexible workflow encapsulates up to 10,000 cells or nuclei per sample together with gel beads into nanodroplets (single-Poisson distribution loading). Up to eight samples can be processed per run allowing for capture of up to 80,000 cells per run. Trained core staff will prepare the libraries from single-cell suspensions submitted by the research investigator. Cells can be suspended in a wide range of media or buffers. 

• 3’ scRNA or 5’ scRNA
• 5’ scRNA and V(D)J (B Cells) or (T Cells) or Both
• V(D)J (B Cells) or (T Cells) or Both 
• V(D)J (B Cells) or (T Cells) or Both and Hashing (Cell surface protein)

All samples are performed by highly trained and experienced laboratory professionals. We follow strict quality procedures to ensure your samples have the best result as possible. Below is this description of a regular sequencing workflow.

10X Genomics Chromium Workflow

1. Creating Project and Reservation
   • Create Project using iLab https://vanderbilt.corefacilities.org/
     • For non-Vanderbilt customers, please sign-up as non-Vanderbilt user
     • Once you are signed in, search for VUMC VANTAGE to request service
   • Reservation Single-Cell arrival time
     • Please email in advance to schedule a date and time when the single-cell will be isolated and arrived. We prefer the last reserve appointment to be at 3 pm, however can make adjustment for a later drop off time
     • Since this is a time sensitive procedure, we ask the estimated arrival time to be 1 hour within the scheduled time. This allows the reagents to thaw before use 
     • For cancellation, please let us know at least 30 minutes before the scheduled time   

2. 10X Sample Preparation Guidelines Recommendations
   • Cell viability needs to be >90%
   • Cell concentration needs to be 750-1200 cells/ul and should be determined via an automated counter. If you do not have access to one, please contact VUMC FLOW CORE for more information 
   • Up to eight samples can be processed per run allowing for capture of up to 80,000 cells per run

3. Single Cell and Spatial Assays

   10x Genomics Single Cell and Visium Spatial Gene Expression Profiling Services

   The 10X Genomics Chromium Platform is a single-cell profiling technology that enables the analysis of large cell numbers at a high capture efficiency (up to 65%). The Chromium Controller and Chromium X Series instruments allow for high-throughput single cell transcriptome, immune profiling and chromatin accessibility analysis in a variety of cell types as well as single nuclei. The flexible workflow encapsulates up to 10,000 (standard assay) or 20,000 (high throughput assay) cells or nuclei together with gel beads into nanodroplets (single-Poisson distribution loading). In singleplex, up eight samples (standard assay) or sixteen samples (high throughput assay) can be processed per run allowing for capture of up to 80,000 or 320,000 cells per run, respectively. Trained core staff will prepare the libraries from single-cell suspensions submitted by the research investigator. Cells can be suspended in a wide range of media or buffers. 

   The Visium Spatial Gene Expression Platform allows for whole transcriptome profiling in both fresh frozen and FFPE tissue sections. Unbiased gene expression information can be obtained from the entire tissue section being analyzed without the need for preselection of areas of interest. Trained core staff have experience sectioning and staining onto Visium Gene Expression slides. Following staining, slides will be imaged and the VANTAGE core will prepare libraries.  

   Current applications offered by VANTAGE include

• Single cell RNA Profiling 
  • Chromium NextGEM 3’ Single Cell Assay
  • Chromium NextGEM 5’ Single Cell Assay
  • Chromium Flex (fixed cell/nuclei) Assay
• Single Cell Immune Profiling
  • Chromium Next GEM 5’Single Cell Assay
  • Bcell and Tcell Immune Repertoire Profiling
  • Cell Surface Protein Profiling
• Single Cell Multiome (ATAC + Gene Expression)
• Single Cell ATAC
• Visium Spatial Gene Expression Profiling

4. QC Check and Balancing

​​​Library Quality Control analysis will be performed by using Qubit and BioAnalyzer to determine the concentration and size bp. Each library that passes the final QC undergoes qPCR using the KAPA library quantification kit and QuantStudio. qPCR allows highly accurate quantification of library molecules that can seed clusters and balance target sample representation during pooling. Multiplexed samples are pooled in equimolar or user-defined ratios to achieve the multiplexing level requested by the user using Illumina MiSeq platform.

5. Sequencing

Final pooling and rebalancing to ensure target sample/cell representation and user defined ratio are achieved. Sequencing will be performed at Paired-End 150 bp on the Illumina NovaSeq 6000.

6. Data Analysis

The resulting data are analyzed with the free Cell Ranger and Loupe Cell Browser software.

• MiSeq
  • Nano: PE-150, PE-250 
  • V2: PE-150, PE-250 
  • V3: PE-300

• NovaSeq 6000

  • SP: PE-50, PE-150, PE-250 
  • S1: PE-50, PE-100, PE-150
  • S2: PE-50, PE-100, PE-150
  • S4: PE-100, PE-150

The nucleic acid QC services include a quantitation and an integrity assessment.  The library QC service includes a quantitation, library profile assessment and a qPCR. We prefer to have at least 12 uL of working volume per sample to allow for a complete Quality Control assessment and sample preparation.  We don’t have a minimum concentration requirement as each protocol is different, but we do ask that the samples not be normalized as the quantitation methods used by different labs can result in very different quantitation results.