Research - Core Facilities - Sequencing Platform

István NAGY
research associate

Dóra LATINOVICS junior research associate
Tünde TOLNAI secretary
Gerda STRIFLER Ph.D. student

SEQUENCING PLATFORM

By integrating the Sequencing Platform of the BAYGEN Institute into BRC HAS the best equipped Sequencing Platform of the region has been implemented. The new platform runs equipment/units of top-quality both in the field of capillary electrophoresis (CE) and Next Generation Sequencing (NGS)

  • 2 units of 3500 Series Genetic Analyzers (Life Technologies) CE instrument with 8 capillary
  • 1 unit of SOLiD 4 (Life Technologies) NGS Instrument
  • 1 unit of SOLiD 5500xl (Life Technologies) NGS Instrument
  • 1 unit of EZ Bead System that is an automated NGS sample preparation system
  • bioinformatics unit equipped with unique sequence analyzing softwares

Capillary Electrophoresis

DNA sequencing

The most precise method used for defining genetic variability is Sanger sequencing. The capillary The 3500 series together with Life Technologies BigDye® Cycle Sequencing Kit provides higher automation, more precise data control and easier usage compared to earlier versions.

The most important parameters of the 3500 series are speed, precision, reproducibility and numerous options to define the length of DNA reading.

During the analysis of samples purified by using the available specific sequencing modules – e.g. use of BigDye® XTerminator™ Purification Kit – the quality of sequences is further improved.

Fragment analysis

The 3500-series is suitable for simultaneous detection of 6 different fluorescent dies thus making possible the increase of multiplexing during fragment analysis which ensures improved quantity of data per run and reduction of expenses per sample.

Next Generation Sequencing (NGS)

Introduction of genomic approach into molecular biology revolutionized experimental biology and made it possible to get acquainted with molecular function of living systems more quickly and thoroughly. Genomics mean the analysis of genome scale that is to extend research to the complete analysis of the certain being or population at DNA-level (structural and metagenomics) or expression-level (gene, microRNS-, protein-expression: functional genomics, expression networks: system biology). Genomic applications play part particularly in understanding the functions of genes which results in 1. a continuous change of approach in basic research both in molecular and cell biology, and 2. exploitable knowledge from the point of view of applied research such as biomedicine, agriculture , forensic, etc.

Genomics shows an unbelievable development in the developed countries of the world; the best example of which is the determination of human genome that started at the beginning of the 90’s and finished in 2001. Then it required more than ten years to determine the sequence of the ~3 billion base pairs while today, only some 11 years later, this can be executed within a couple of weeks thanks to a revolutionary new technique. In western countries genomics has already broke into everyday medicine: several companies have been founded in the US which make genomic services accessible to the private section as well.

Next Generation Sequencing - launched on the market in 2005 - makes technical implementation of nowadays’ leading and complete genomic-level analysis, the so called ultra-high throughput possible. By way of these instruments we are able to analyse diversified biological samples from the analysis of phylogenetic tree of microbes, through sequencing following immunoprecipitation of chromatin (ChIP-Seq), to the analysis of gene’s copy number (detailed below).

The SOLiD systems (S4 and 5500xl) operated by our Sequencing Platform are revolutionary new genetic analysing platforms which make parallel sequencing of clonally amplified DNA fragments attached to the surface on magnetic beads possible. The ultra-high output and unique precision coupled with wide application flexibility guarantee an outstanding system.


Main parameters of the system


Output >60GB/course >600 million sequence-tag per run >225GB/course >1,5 billion sequence-tag per run
Reading length 50 base pairs in case of fragment library
2x50 base pairs in case of „mate-paired” library
75 base pairs in case of fragment library
2x75 base pairs in case of „mate-paired” library
Insert size from 600 bp to 10000 bp in case of „mate-paired” library
Sequencing chemistry ligation based; 2-bases encoding system
repeatable ligation steps
Precision >99,94% thanks to the two-base encoding
Multiplex experiments by way of chemical barcoding – with reducing unit costs
Starting sample from blood, BAC, plasmid, fosmid, tissue, cells (even from few cells), DNA or cDNA isolated from PCR product
total RNA or purified mRNA/microRNA populations
Starting sample quantity fragment DNA library: 20ng – 2μg (cc >400ng/μl)
„mate-paired” DNA library: 5μg - 20μg (cc >400ng/μl)
in case of total and mRNA: >5μg (cc >400ng/μl; RIN > 8.0)
in case of microRNS: >100ng

Application fields

  • de novo sequencing (sequencing of species of unknown genome, and subsequent determination of their nucleotid sequence (genome),
  • resequencing (sequencing of species of known reference genome, e.g. phylogenetic systematization of bacteria, identification of sequence variations and SNPs),
  • comparative -, meta-, population genomics
  • gene-expression (RNA-Seq; Digital Gene Expression Profiling, DGE),
  • haplotype definition,
  • microRNA-expression, -identification,
  • sequencing following chromatin immunoprecipitation (ChIP-Seq; identification of transcription factor binding sites; finding active or passive chromatin),
  • DNA methylation analysis (Meth-Seq).

Selected publications

Hunyadkürti J., Feltóti Zs., Horváth B., Nagymihály M., Vörös A., McDowell A., Patrick S., Urbán E. and Nagy I. (2011) Complete Genome Sequence of Propionibacterium acnes Type IB strain 6609. Journal of Bacteriology vol: 193, issue 17; 4561-4562

Horváth B., Hunyadkürti J., Vörös A., Fekete C, Urbán E., Kemény L. and Nagy I. (2012) Genome Sequence of Propionibacterium acnes Type II strain ATCC11828. Journal of Bacteriology vol: 194, issue 1; 202-203.

Kriszt B., Táncsics A., Cserháti M., Tóth Á., Nagy I., Horváth B., Nagy I., Tamura T., Kukolya J. and Szoboszlay S. (2012) De novo genome project of the oil degrader Rhodococcus pyridinivorans AK37. Journal of Bacteriology vol : 194, issue 5; 1247-1248.

Csepregi K., Valasek A., Pénzes Á., Tóth Z., Kiss É.Í., Kerepesi I., Horváth B., Nagy I. and Fekete C. (2012) Draft Genome Sequence of an Efficient Antibiotics Producing Industrial Strain (SZMC 14600) of Saccharomonospora azurea. Journal of Bacteriology vol : 194, issue 5; 1263.

Vörös A., Horváth B., Hunyadkürti J., McDowell A., Barnard E., Patrick S. and Nagy I. (2012) Complete genome sequence of three Propionibacterium acnes isolates from the type IA2 cluster. Journal of Bacteriology vol: 194, issue 6; 1621-1622.

Cserháti M, Kriszt B., Szoboszlay S., Tóth Á, Szabó I., Nagy I., Horváth B., Nagy I. and Kukolya J. (2012) De novo genome project of Cupriavidus basilensis strain OR16. Journal of Bacteriology vol: 194, issue 8; 2109-2110.

McDowell A., Hunyadkürti J., Horváth B., Vörös A., Bernard E., Patrick S. and Nagy I. (2012) Draft Genome Sequence of an Antibiotic Resistant Propionibacterium acnes Strain PRP-38 from the Novel Type IC Cluster Journal of Bacteriology vol: 194, issue 12; 3260-3261.

McDowell A., Barnard E., Nagy I., Gao A., Tomida S., Li H., Eady A., Cove J., Nord C.E. and Patrick S. (2012) An Expanded Multilocus Sequence Typing Scheme for Propionibacterium acnes: Investigation of 'Pathogenic', 'Commensal' and Antibiotic Resistant Strains PLoS One vol: 7, issue 7; e41480.

Gór B., Balla A., Tukacs E., Nagy I., and Török Z. (2012) A novel high accuracy algorithm for reference assembly in colour space. International Journal of Biometrics and Bioinformatics vol: 6, issue 4; 92-104.