g e n o m i c s s i m p l i f i e d

Introduction Results

• Human mitochondria has a 16.5 kb genome which is essentially maternally inherited and does not go through recombination.

• However there are a few hundred copies of Mitochondria in each cell, with mutations carried in some of the copies.

• Mitochondrial gene mutations are implied in aging, tumor suppression or susceptibility to diseases like cancers, neurodegenerative diseases, muscle disorders and infertility.

• Mitochondrial sequencing also reveals mutation frequency estimation for population mapping, human migration pattern, population divergence and Forensics analysis.

This poster presents the methodology and results of a custom assay – MitoKitGT, designed for sequencing of mitochondrial DNA (mtDNA) from sheared, fixed and even ancient DNA. It uses home-brew primers to amplify the mtDNA following by sequencing on the Ion Torrent PGM platform and a custom analysis algorithm to call the variants. MitoKitGT can be used for forensic investigations and population studies, for diagnosis of diseases arising due to mitochondrial defects, and for detecting heteroplasmic mutations that are particularly important in ageing research.

Applications of Mitochondrial sequencing

Snapshot of variant calls

Coverage of the Mitochondrial Genome

Advantages of MitoKitGT

Technology challenges to develop a high sensitivity assay for Mitochondria

• Traditional methods of diagnosing mitochondrial defects are based on histochemical and biochemical assays. A sequencing-based assay is more precise and can detect specific mutations.

• However, isolation of enriched mtDNA from the genomic DNA is a challenge.

• Variable clinical samples such as FFPE samples with sheared DNA makes the mtDNA isolation difficult.

• Inherent heteroplasmy across multiple copies of mitochondria in a cell demands the use of a sensitive assay.

• MitoKitGT overcomes all these challenges to provide a fast, sensitive, and cost-effective assay that works with a variety of samples.

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Conclusion

Experimental Methodology

Deepti Saini*, Nandita Mullapudi*, Althea F, Bharath Kumar E, Ramya K, Manoharan K, Sanchita Anand, Ramesh G, Priya G, Rushiraj M and Sudha N Rao ** *Authors contributed equally ** corresponding Author sudha.rao@genotypic.co.in Genotypic Technology, Bangalore, India. www.genotypic.co.in

MitoKitGT- Mitochondrial Genome sequencing by NGS for Molecular diagnostics.

Workflow of MitoKitGT

Variant alignment report Ion Torrent

Tissue from patient or

biopsy sampleDNA Amplicon pools Multiplexed

Amplicon poolsIon tagging and

barcoding

Match to curated datasets for pathological mutations

Custom Scripts..

Clinical disorders that are caused by mutations in mitochondrial DNAMitochondrial DNA Disorder Clinical phenotype mtDNA genotype Gene Status Inheritance Reference

Kearns–Sayre syndromeProgressive myopathy, ophthalmoplegia, cardiomyopathy

A single, large-scale deletion Several deleted genes Heteroplasmic Usually sporadic 61,158

CPEO Ophthalmoplegia A single, large-scale deletion Several deleted genes Heteroplasmic Usually sporadic 61, 64

Pearson syndrome Pancytopoenia, lactic acidosis A single, large-scale deletion Several deleted genes Heteroplasmic Usually sporadic 65

MELASMyopathy, encephalopathy lactic acidosis, stroke-like episodes

3243A>G; 3271T>C TRNL1 Heteroplasmic Maternal 159

Individual mutations ND1 and ND5 Heteroplasmic Maternal 160, 161

MERRF Myoclonic epilepsy, myopathy 8344A>G; 8356T>C TRNK Heteroplasmic Maternal 162

NARPNeuropathy, ataxia, retinitis pigmentosa

8993T>G ATP6 Heteroplasmic Maternal 163

MILSProgressive brain-stem disorder

8993T>C ATP6 Heteroplasmic Maternal 67

MIDD Diabetes, deafness 3243A>G TRNL1 Heteroplasmic Maternal 164

LHON Optic neuropathy 3460G>A ND1Hetero- or homoplasmic

Maternal 165

11778G>A ND4Hetero- or homoplasmic

Maternal 62

14484T>C ND6Hetero- or homoplasmic

Maternal 166

Myopathy and diabetesMyopathy, weakness, diabetes

14709T>C TRNEHetero- or homoplasmic

Maternal 167,168

Sensorineural hearing loss Deafness 1555A>G RNR1 Homoplasmic Maternal 55

Individual mutations TRNS1Hetero- or homoplasmic

Maternal 169,170

Exercise intolerance Fatigue, muscle weakness Individual mutations CYB Heteroplasmic Sporadic 68

Fatal, infantile encephalopathy;Leigh/Leigh-like syndrome

Encephalopathy, lactic acidosis

10158T>C; 10191T>C ND3 Heteroplasmic Sporadic 66

Nat Rev Genet. 2005 May; 6(5): 389–402.

• MitoKitGT has been successfully applied in SNP variant calling in control DNA samples as well as some ancient degraded DNA samples.

• This assay has practical applications in aging studies, testing susceptibility to diseases like cancers, neurodegenerative diseases, muscle disorders and infertility.

• Other potential uses of MitoKitGT include mutation frequency estimation for evolution studies, population mapping, human migration pattern, population divergence and Forensics analysis.

Precise, rapid and cost-effective sequencing of human mtDNA from any sources using MitoKitGT can be availed from the high-throughput genomics laboratory of Genotypic Technology.

#2/13, Balaji Complex, Poojari Layout, R.M.V 2nd Stage,80 Feet Road, Bangalore - 560094. INDIA . Tel: +91 80 40538200 | Fax: +91 80 40538222, International Hotline: +1-(215) 253 4495, Email: genomics@genotypic.co.in Website: http:\\www.genotypic.co.in, www.facebook.com/genotypic

Genotypic Technology Pvt Ltd

Agilent Certified

Services ProviderMicroarray-basedGenomics Analysis

Agilent Certified

Services ProviderTarget EnrichmentSystem

TM

Ion AmpliSeq ™

ion torrent certifiedserviceprovider

MitoKitGT Other MethodsPCR based for small amplicons PCR based for large amplicons

No library preparation required before sequencing Library preparation required

Works with fresh and archived samples (intact and degraded DNA)

May work with different types of input sample

Can be sequenced on multiple platforms Platform specific library preparation

Simple two step procedure from sample to sequencing

Multi step protocols from sample to library preparation for sequencing

Fast Time consuming

Cost effective Comparatively expensive