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
|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)|
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|
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|
|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.
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.
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.
|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)|
|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|
|SNV & InDel detection|
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.
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.
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.
|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™