Genomics Facility currently houses two instruments i.e. DNA Sequencer: ABI 3500 Genetic Analyzer and Droplet Digital PCR (Bio-Rad).
1. DNA Sequencer: ABI 3500 Genetic Analyzer
3500 Genetic Analyzer is a fluorescence based 8-capillary sequencing instrument which is based on Sanger DNA sequencing. The system is configured with 50 cm array and an integrated software for instrument control, data collection, quality control, base-calling and size-calling of samples. We use BigDyeTM Terminator v3.1 cycle sequencing kit, POP-7TM, and BigDye XterminatorTM for short-to-long-read sequencing. The system has a varied range of applications like microbial sequencing, targeted DNA sequencing, epigenetic analysis, pathogen analysis, genetic disease research, SNP analysis and biomarker analysis. Currently, a range of cloned DNA, PCR products and BAC DNA are analyzed with a read length of up to 1000 bp.
2. Droplet Digital PCR: ddPCRTM (Bio-Rad)
ddPCRTM is an advanced version of PCR that provides simultaneous clonal amplification and fluorescence-based quantification of nucleic acids. A typical reaction in ddPCRTM is partitioned into several thousands of water-in-oil droplets where individual reactions are carried out providing absolute, precise and reliable quantification. The system includes: i) QX 200TM Droplet Generator; ii) T100TM Thermal Cycler; iii) PX1TM PCR Plate Sealer and iv) QX 200TM Droplet Reader. The reaction utilizes nucleic acids, primer sets, fluorescent probes (TaqMan probes with FAM and HEX or VIC), and proprietary supermix for droplet generation. The QX 200TM Droplet Generator generates approximately 20,000 nanoliter-sized uniform droplets which are transferred to a 96-well plate for clonal amplification. After the reaction, the droplets are read in the QuantasoftTM software bundled in QX 200TM Droplet Reader that counts PCR-negative and PCR-positive droplets and provides an initial DNA concentration of target molecule by statistical analysis in terms of copies/µL. ddPCRTM has demonstrated usage in the detection of rare DNA targets, determination of copy number variations and measurement of gene expression levels.
3. Human Cell Line Authentication
Cell line work suffers from dual issues, viz contamination with Mycoplasma or with other cell lines. As per recent study, cross-contamination of cell lines is being observed at an unacceptably higher levels - up to 18% percent. STR profiles aid in ensuring both quality and integrity of human cell lines, that are employed to advance biomedical research and produce quality biologicals. Cell line authentication (CLA) is required for following reasons: several key journals demand CLA; project granting agencies seeks proof of cell line authenticity; cell line misidentification, cross-contamination and associated financial burden due to unreliability of data generated. Sample could be either frozen cell pellet or gDNA. Genomic DNA extracted from samples using DNeasy blood and tissue kit (Qiagen) will be subjected to STR profiling with GenePrint 24 system (Promega). Process involves multiplex PCR with fluorescently labeled primers, Capillary Electrophoresis using AB3500 Genetic analyzer and raw data analysis using GeneMapper software 5. STR profile for 23 loci plus amelogenin genotype will be provided. However, for Human CLA, 8 loci STR profile, as recommended by ATCC Standards Development Organization (Ref: ASN-0002: Authentication of Human Cell Lines: Standardization of STR Profiling) will be analysed and reports generated. STR analysis results from publicly available STR database, namely, DSMZ (or ATCC) along with Electropherograms and expert interpretation containing report are provided.
Sample Requirement: Human Cell lines to be tested could be provided as frozen cell pellet with cell density being 1-2 million cells/ml; If gDNA (samples should strictly be eluted in nuclease free water (not DEPC treated)/TE Buffer; Desirable Conc & Vol: atleast 50ng/µl; 10µl volume) isolated from cultures is to be provided, then We recommend using DNeasy Blood & Tissue Kit (Qiagen; Cat#69504), in line with our SOPs. Properly labelled Gel picture with vol of sample loaded, should be emailed, in case of gDNA samples; ATPC does not accept cultures infected with HIV or BSL 3 or 4 agents as well as cultures containing any
carcinogens, for Human CLA). The turnaround time for results is 8 business days.
4. Mycoplasma Testing
Mycoplasma are a group of bacteria lacking cell wall and are placed under the class Mollicutes; They are well known to contaminate cell cultures and influence the physiology of the cells under study. Twenty one percent secondary source cell lines (cell lines that were procured from non-repositories, which forego cell-line authentication and Mycoplasma contamination check) were found to be positive for Mycoplasma, as per recent study(PMID:27870004; study by Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Germany). Mycoplasma, due to their lack of cell wall, can escape filter sterilization; Mycoplasmas are known to occur at high titres with no effect on viability, resulting in non-detection of these detrimental contaminants. In order to ensure, reliable lab data generation, frequent testing for Mycoplasma contamination is suggested: upon receiving new cell or virus cultures plus prior to each cryopreservation step; Monthly testing is recommended for continuous cell lines. PCR offers the rapid way to screen these common cell culture contaminants. We employ a PCR method, that uses primers targeting a conserved region within the 16S rRNA gene, to detect Mycoplasma contamination in cell culture supernatant. Our SOP includes appropriate positive control and negative control. The method used detect common contaminating Mollicutes, which includes Acholeplasma laidlawii, Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycoplasma orale, Mycoplasma pneumoniae, Mycoplasma gallisepticum, Mycoplasma synoviae and Spiroplasma citri.
Sample Requirement: 500µl of Cell culture supernatant (Samples must be cultured in the absence of antibiotics known to target Mycoplasma for days [minimum three sub-cultures or 2 weeks] to maximize detection sensitivity; Samples are to be drawn from cultures that have reached 90-100% confluence; Older cell cultures might have accumulated substances that are inhibitory to PCR. Hence provide us only fresh culture based samples for testing) / gDNA. The turnaround time for results is 2 business days. Gel picture will be provided.
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