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NGS Services

MedGenome offers an end-to-end solution for next-generation sequencing as a service for a wide range of sample types (DNA, RNA and specialized samples like T cells and B cells) as shown in Figure 1. The user has flexible options to use our services from end-to-end or simply request services at any step of the workflow. To learn more about our services click on the tabs in the workflow below.

Figure 1 : Workflow of the NGS Services provided at MedGenome for a wide range of sample types. Shown in this diagram, we highlight the steps taken to validate successful sample preparation, library generation, sequencing and data analysis for successful completion of a project. Click on each tab to learn more about the options available, steps followed and the QC reports generated.

Sample QC : Qubit, Gel and TapeStation

Library QC : TapeStation Bioanalyzer and Qubit

Data QC : Read orientation, quality distribution, base distribution and GC distribution for all reads

Overview of NGS services workflow

Sample Type DNA (WGS/WES/Targeted) WTS/Targeted/Small RNA (Transcriptome)
Extracted Nucleic Acid Minimum: 200 ng (WGS/WES) 50 ng (Targeted) Minimum: 250 pg (DV200 > 25%)
Frozen cell pellets Minimum 1 x 106 cells (ship on dry ice) Minimum 1 x 106 cells (ship on dry ice)
Frozen tissues 30 mg frozen tissue or 20 mg stabilized tissue (ship on dry ice) 30 mg frozen tissue or 20 mg stabilized tissue (ship on dry ice)
FFPE slides 3-6 slides (10 μM thick) 250 mm2 surface area 3-8 slides (10 μM thick) 250 mm2 surface area
Curls 5 curls (10 μM thick) 8 curls (10 μM thick)
Single-cell Fresh or cells in sample prep agent Fresh or cells in sample prep agent

Lower RNA & DNA amounts can be used for special projects

Service Type Kit Used
RNA (whole transcriptome) Transcribed exonic regions Small RNA Illumina TruSeq RNA, Takara SMARTer Pico V2, Illumina TruSeq RNA, Access, Stranded (no capture probes used)
Whole exome (WES) Agilent SureSelect Human All Exon V5/V6 kitAgilent SureSelect XT MouseAgilent SureSelect + UTR X5TruSeq Exome
Whole genome (WGS) Illumina TruSeq DNA PCR-Free kit10X WGS Chromium linked read prepIllumina TruSeq NANO KAPA (no capture probes used)
MethylSeq Agilent Sure Select XT Methyl-Seq Target Enrichment kit
TCR sequencing 10X V(D)J, SMARTer TCR Profiling
Panel sequencing Custom design (Nimblegen, IDT)
Sample Type Illumina Sequencing Platform
WGS/WES/Targeted Illumina NovaSeq 6000, HiSeq 2500, HiSeq X
Whole-transcriptome profiling Illumina NovaSeq 6000, HiSeq 2500, MiSeq
TCR profiling Illumina MiSeq, NovaSeq 6000 (high throughput single-cell)

TCR Sequencing

Whole Genome / Whole Exome Sequencing

Whole Genome and Exome Sequencing using NGS have been widely accepted to speed up and reduce the cost of sequencing genomes for basic research as well as use of genomic data for a wide range of applications : genome wide association studies for complex diseases, variant calling to identify clinically actionable mutations and other specialized areas like identification of mutations that accumulate and give rise of tumor neo-antigens and shared antigens. At MedGenome we routinely perform Whole Genome Sequencing and Exome Sequencing and analyses using best practices.

Table 1 : Sample types processed for WGS/WES at MedGenome.

Sample Type DNA (WGS/WES/Targeted)
Extracted Nucleic Acid Minimum: 200 ng (WGS/WES) 50 ng (Targeted)
Frozen Cell Pellets Minimum 1 x 106 cells (ship on dry ice)
Frozen Tissues 30 mg frozen tissue or 20 mg stabilized tissue (ship on dry ice)
FFPE Slides 5 slides (10 μM thick) 250 mm2
Curls 5 curls (10 μM thick)
Single-cell Fresh or cells in sample prep agent

Table 2: Deliverables for analyses of WGS/WES data.

Standard Analysis Offering Advance Analysis Offering
Data QC (Read orientation, quality distribution, base distribution and GC distribution for all reads) CNV analysis
Alignment to the reference genome Pathway analysis
Coverage analysis Deep variant annotation
Variant calling (germline/somatic calling) Mutational burden analysis
Variant Annotation Variant interpretation

WGS germline calling validation

Total number of SNPs and Indels predicted using different flavour of GATK on two different datasets

Dataset Germline Variant Caller #of SNPs #of Indels Total
Gold Standard GiAB 3,193,799 391,621 3,585,420
Illumina HiSeq X GATK - Lite - 2.3.9 -UC 3,191,291 380,768 3,572,059
Sentieon - 3.5 -HC 3,213,528 393,803 3,607,331
Sentieon - 3.5 -UC 3,230,950 379,220 3,610,170
GATK - 3.6 -HC 3,184,106 388,192 3,572,298
GATK - 3.6 -UC 3,191,477 376,688 3,568,165
Illumina HiSeq 2500 PublicData (NIST) GATK - Lite - 2.3.9 -UC 3,198,322 383,952 3,582,274
Sentieon- 3.5 -HC 3,215,682 397,577 3,613,259
Sentieon - 3.5 -UC 3,242,040 386,397 3,628,437
GATK - 3.6 -HC 3,186,676 393,300 3,579,976
GATK - 3.6 -UC 3,198,478 383,917 3,582,395
  • Sensitivity for SNV detection of our pipeline exceeded 99% at 30X-50X coverage and was over 99% for the detection of Indels at 50X coverage.

SNP Metrics
InDel Metrics

Figure 2 : Validation of WGS/WES pipelines using Gold Standard datasets

  • Results from our analysis meet industry standards and FDA challenge results.

We have recently added the 10X Chromium platform to our offerings and can perform 10X linked reads Whole Genome Sequencing, and analysis as well as de-novo analysis using the 10X software solutions.

Whole Transcriptome Sequencing

Next generation sequencing enables gene expression analysis from normal and disease tissues with high-sensitivity. Profiling of coding and non-coding RNAs is important in revealing molecular mechanisms of development and disease. With the several options that exist for gene expression profiling, it is also possible to perform whole transcriptome profiling ranging from a single-cell to thousands of cells in a sample. In addition, current methodologies allow for profiling of transcriptomes from intact RNA and RNA degraded due to the storage and isolation conditions (such as FFPE processed tissue blocks, cell-free nucleic acids etc). At MedGenome we offer solutions, for performing gene expression analysis from a wide-range of sample types. Shown in table 1 is a range of sample types that we process for whole transcriptome profiling.

Sample requirements and library prep information

Service Sample Requirements Library Kit Used
RNA (whole transcriptome) Minimum = 250 pg (DV > 25) Minimum: 50 ng (RIN < 5) Single-cell solutions available TruSeq RNA, SMARTer Pico v2, TruSeq Stranded (No capture probes used)

RNA analysis deliverables

Standard Analysis Offerings
Data QC (read orientation, quality distribution, base distribution and GC distribution for all reads)
Alignment to the reference genome & transcriptome
Gene and isoform expression estimation in RPKM / read count per gene
Statistical analysis including p-values and coefficient of variance (CV) between replicates
Gene/Transcript expression levels in Excel
Differential expression comparison
Advanced Analysis Offerings
Gene fusion detection
Wiggle file for gene expression visualization on genome browser
Pathway analysis
SNV & InDel detection
Splicing analysis

In addition to the NGS library preparation solutions, we offer extensive informatics solutions for whole-transcriptome profiling that ranges from standard gene expression analyses to advanced analyses of splicing, pathways, visualization and tertiary analyses to reveal heterogeneity and cell types in a population as well tumor microenvironment analyses. In addition to that we have solutions using machine learning approaches that utilize gene expression data to predict biomarkers and classify tissue types and diseases.

TCR Sequencing

T-cells are the core components of our adaptive immune system. Once activated, they can directly kill cells that are foreign (cytolytic T-cells) or perform helper function (helper T-cells) to activate B-cells to make antibodies against foreign antigen. The activation of T-cells involve recognition of MHC-peptide complex by the T-cell receptors (TCR). Humans carry >109 T-cells, each expressing a unique TCR. This highly diverse repertoire of T-cells has the ability to recognize peptides originating from foreign elements such as invading pathogens and cancer cells. Each TCR recognize peptides in complex with MHC proteins presented on the surface of antigen presenting cells. Productive T-cell activation results in the clonal expansion of a specific T-cell and this expansion can be accurately determined by TCR sequencing.

TCR profiling holds great potential not only for understanding the mechanisms of development of the normal immune response, but also in providing insights into disease mechanisms and development of new therapeutics and treatment modalities in infectious diseases, autoimmunity and in immuno-oncology. However identification of all potential clonotypes in a diverse repertoire of TCRs requires sensitive methods of detection. Next-generation sequencing (NGS) technologies have recently enabled accurate detection of TCRs, and in combination with other assays allow for the assessment of the TCR repertoire in patients- which in turn is a proxy for patient prognosis and response. At MedGenome, we provide TCR repertoire profiling using bulk input (from cells, RNA and FFPE tissue) using the SMARTer® TCR Profiling Kit (Takara Bio USA Inc) and single-cell inputs using the SMARTer single-cell TCR Profiling kit and the Chromium Immune Profiling solutions (10X Genomics). To learn more about the different applications and sample data generated using TCR Seq at MedGenome read the white paper.

Major Applications of TCR Repertoire Profiling

Figure 1. Overview of major applications of TCR Repertoire profiling in a) Therapeutics application: TCR Sequencing of lymphocytes to identify antigen specific TCRs to generate engineered T cells for adoptive cell therapy b) Diagnostics assay for clonality for auto immune disorders c) Treatment monitoring: to identify biomarkers of response.

Overview of MedGenome Offerings:

Name of offering Input type Amount of material neeeded Analysis method Information Obtained
Single cell Immune profiling (10X Genomics) isolated cells Single-cells (Human, Mouse) Loupe browser CDR3, α/β pairing and clonotypes V(D)J sequences
SMARTer TCR Profiling Kit (Takara Bio USA) isolated cells or RNA 10 ng-3 ng/50-10,000 cells (Human, Mouse) MiXCR CDR3, V (D)J sequences α/β pairing(from SC Kit)

In addition to the TCR Seq services, MedGenome offers advanced informatics solutions for TCR binding based prediction of immunogenic neoepitopes for cancer vaccine development using OncoPeptVAC™