Pracownie usługowo-badawcze

Dr Robert Gromadka

Środowiskowa Pracownia Sekwencjonowania i Syntezy DNA

Zakres usług i badań

Our facility provides services for DNA/RNA sequencing and oligonucleotide synthesis. Our sequencing technology portfolio includes first-generation sequencing (classic Sanger and fragment analysis), second-generation sequencing (next-generation sequencing) using Illumina instruments, and third-generation long-read nanopore direct DNA/RNA sequencing. Our facility maintains a fully equipped laboratory and expert staff who perform various sequencing applications, from sample preparation to data analysis.

Contact person

Robert Gromadka (PhD), email: robert@ibb.waw.pl, tel.: +48 22 592 3402

Ordering and results

http://oligo.ibb.waw.pl/

Usługi

Opis usług

The main aims of our facility are the massive sequencing of genomic DNA/RNA and functional genomics. Our facility also provides the synthesis of oligonucleotides for sequencing, polymerase chain reaction (PCR), mutagenesis, and gene synthesis. We are a co‑executor of grants based on sequencing techniques. Additionally, we provide services to other laboratories at IBB and many other institutions in Poland. Our facility is equipped with a large portfolio of high-throughput sequencers for classic sequencing using Sangers' method (ABI3730 and ABI3730xl), next-generation sequencing using MiSeq and NextSeq 550 instruments, and third-generation long-read sequencing using MinION and GridION. Additionally, through a collaboration with the Mother and Child Institute in Warsaw, we have access to an Illumina NovaSeq 6000 ultra-deep sequencing system. As a result, depending on specific requirements, we can provide sequencing throughput with a low or high output of data. In addition to sequencing applications, we perform sample preparation and quality control assessments for each of our platforms. Each sequencing service includes data processing, quality control, and data delivery. We deliver high-quality, filtered sequence data and associated quality scores. We also provide comprehensive data analysis based on customer demands. Our facility focuses on the sequencing of small genomes (bacteria and viruses) and medium-size genomes of eukaryotes (fungal and algal genomes). Based on strong expertise in long-read sequencing, the generation of complete genomes with high consensus accuracy is one of our great advantages. We also offer targeted amplicon sequencing for microbiome analysis, such as selected variable regions of 16S rRNA or fungal fungal the internal transcribed spacer (ITS) fragments. To test changes in expression levels and discover novel transcripts or single-nucleotide polymorphisms (SNPs), we can conduct transcriptome and metatranscriptome sequencing (RNA sequencing). Additionally, using the 10x Genomics Chromium platform to capture hundreds to tens of thousands of single cells, we can provide studies of gene expression profiles or changes in the genome of a single cell.

Aparatura

MinION and GridION Oxford Nanopore Technologies – Real-time DNA and RNA sequencer of 3rd generation for 1 or 6 parallel independent flow cells respectively.

Parameters of single flow cell: 1 min – 72 hour run time, up to 30 Gb (theoretical maximum up to 50 Gb), 512 channels. Nanopores read the length of DNA or RNA from short to ultra-long (longest >4 Mb). Suitable applications include:

Whole genomes/exomes

– Metagenomics

– Targeted sequencing

– Whole transcriptome (cDNA)

– Smaller transcriptomes (direct RNA)

– Multiplexing for smaller samples

 

NextSeq 550 Illumina – Sequencer of 2nd generation.

Sequencing Performance Parameters

NextSeq 550 System High-Output Kit* NextSeq 550 System Mid-Output Kit*
Read Length Total Time Output Read Length Total Time Output
2 × 150 bp 29 hrs 100–120 Gb 2 × 150 bp 26 hrs 32.5–39 Gb
2 × 75 bp 18 hrs 50–60 Gb 2 × 75 bp 15 hrs 16.25–19.5 Gb
1 × 75 bp 11 hrs 25–30 Gb

*Install specifications based on Illumina PhiX control library at supported cluster densities (between 129 and 165 k/mm² clusters passing filter). Actual performance parameters may vary based on sample type, sample quality, and clusters passing filter. All NextSeq 550 kits are paired-end capable.

†Total time includes cluster generation, sequencing, and base calling on a NextSeq 550 System enabled with dual-surface scanning.

Reads Passing Filter

  NextSeq 550 System High-Output Kit NextSeq 550 System Mid-Output Kit
Single Reads Up to 400 Million Up to 130 Million
Paired-End Reads Up to 800 Million Up to 260 Million

Quality Scores††

NextSeq 550 System High-Output Kit NextSeq 550 System Mid-Output Kit
> 75% bases higher than Q30 at 2 × 150 bp > 75% bases higher than Q30 at 2 × 150 bp
> 80% bases higher than Q30 at 2 × 75 bp > 80% bases higher than Q30 at 2 × 75 bp
> 80% bases higher than Q30 at 1 × 75 bp

††A quality score (Q-score) is a prediction of the probability of an error in base calling. The percentage of bases > Q30 is averaged across the entire run.

Key Applications and Methods:

– amplicon sequencing

– whole genome sequencing

– whole transcriptome sequencing

– targeted DNA resequencing

– environmental DNA and RNA sequencing

 

MiSeq Illumina – Sequencer of 2nd generation.

Sequencing Performance Parameters

MiSeq Reagent Kit v2 MiSeq Reagent Kit v3
Read Length 1 × 36 bp 2 × 25 bp 2 × 150 bp 2 × 250 bp 2 × 75 bp 2 × 300 bp
Total Time* ~4 hrs ~5.5 hrs ~24 hrs ~39 hrs ~21 hrs ~56 hrs
Output 540–610 Mb 750–850 Mb 4.5–5.1 Gb 7.5–8.5 Gb 3.3–3.8 Gb 13.2–15 Gb
MiSeq Reagent Kit v2 Micro MiSeq Reagent Kit v2 Nano
Read Length 2 × 150 bp 2 × 250 bp 2 × 150 bp
Total Time* ~19 hrs ~28 hrs ~17 hrs
Output 1.2 Gb 500 Mb 300 Mb

* Total time includes cluster generation, sequencing, and base calling on a MiSeq System enabled with dual-surface scanning.

Reads Passing Filter**

MiSeq Reagent Kit v2 MiSeq Reagent Kit v3 MiSeq Reagent Kit v2 Micro MiSeq Reagent Kit v2 Nano
Single Reads 12-15 million 22–25 million 4 million 1 million
Paired-End Reads 24–30 million 44–50 million 8 million 2 million

** Install specifications based on Illumina PhiX control library at supported cluster densities (865‑965 k/mm2 clusters passing filter for v2 chemistry and 1200-1400 k/mm2 clusters passing filter for v3 chemistry). Actual performance parameters may vary based on sample type, sample quality, and clusters passing filter.

Quality Scores†

MiSeq Reagent Kit v2 MiSeq Reagent Kit v3
> 90% bases higher than Q30 at 1 × 36 bp > 85% bases higher than Q30 at 2 × 75 bp
> 90% bases higher than Q30 at 2 × 25 bp > 70% bases higher than Q30 at 2 × 300 bp
> 80% bases higher than Q30 at 2 × 150 bp
> 75% bases higher than Q30 at 2 × 250 bp

† A quality score (Q-score) is a prediction of the probability of an error in base calling. The percentage of bases > Q30 is averaged across the entire run.

Key Applications and Methods:

– small genome sequencing

– targeted DNA sequencing

– 16S metagenomics sequencing

– miRNA and small RNA sequencing

 

ABI3730 and ABI3730xl DNA Analyzers Thermo Fisher Scientific – Sequencer of 1st generation (Sanger).

The 48- and 96-capillary sequencer respectively.

Specification:

– capacity: 16 sample plates (96-well or 384-well)

– capillary Length: 36 cm or 50 cm

– optics: In-capillary detection by dual-side illumination, Backside-thinned CCD for maximum optical sensitivity, Multiple Filter Sets: Z, E, G5, G5-RCT, Any4Dyeset, Any5Dyeset.

– polymer: POP-7

Perform a wide variety of sequencing and fragment analysis applications including resequencing, microsatellite analysis, AFLP, LOH, SSCP, SNP screening and SNP validation

 

Dr. Oligo 96 Oligo Synthesizer Biolytic Lab Performance Inc. – oligonucleotide synthesizer.

Oligonucleotides synthesized in one run 1-96 in scales 40 and 200 nMole. Oligonucleotides produced with the Dr. Oligo line of DNA synthesizers can be used for Oligonucleotide and Gene synthesis, PCR / RT-PCR, siRNA / RNAi, miRNA, CRISPR Cas9, DNA / RNA sequencing, Gene Construction, Reverse Transcription or Hybridization.

 

10x Chromium Controller 10x Genomics – Single Cell DNA/RNA Sequencing Library construction.

The Chromium Controller is designed to automate parallel sample partitioning and molecular barcoding at the single cell level. The controller allows for single cell analysis of CNV, gene expression, immune profiling, and ATAC. We can process between 1–8 samples in one run, loading up to 10,000 cells per sample.

 

FemtoPulse System Agilent – automated pulsed-field capillary electrophoresis system.

The FemtoPulse system is ideal for long-read NGS QC, gDNA, small RNA, or cfDNA analysis from low concentration samples. It has the ability to separate high molecular weight DNA up to 165 kb and detect nucleic acids down to 50 fg/µL input concentration. Is equipped with 12-Capillary Array for use with the Genomic DNA 165 kb kit, the Ultra Sensitivity NGS kit (1-6000 bp), the Ultra Sensitivity RNA kit, and the 55 kb BAC kit. It has a separation or effective length of 22 cm, with an overall length of 40 cm.

 

LightCycler 96 Roche – real-time PCR instrument.

The LightCycler 96 instrument is a real-time PCR system for rapid cycling in 96-well plate format. System applications include: absolute and relative quantification, melting curve analysis and endpoint genotyping.

 

OT-2 pipetting robot Opentrons – pipetting workstation.

Liquid handling including pipetting, dilution, dispensing and integration, PCR and sequencing reaction cleanup, cell-based assays, normalization, cherry picking, and more. Is equipped with single and 8-channel pipettes, thermocycler module, temperature module and magnetic module.

Współpraca

  • Dr hab. Tomasz Jagielski, Department of Medical Microbiology, University of Warsaw
  • Dr hab. Izabela Sitkiewicz, Center for Population Diagnostics, PORT Polish Center for Technology Development, Wrocław
  • Dr hab. Anna Skoczyńska, Department of Epidemiology and Clinical Microbiology, National Medicines Institute
  • dr hab. Dariusz Plewczyński, Laboratory of Functional and Structural Genomics, CeNT, University of Warsaw
  • dr hab. Brygida Ślaska – Department of General and Molecular Genetics, University of Life Sciences, Lublin

Klienci

  • Instytut Biologii Doświadczalnej im. M. Nenckiego
  • Instytut Biologii Medycznej PAN Łódź
  • Instytut Biotechnologii i Antybiotyków w Warszawie
  • Instytut Biocybernetyki i Inżynierii Biomedycznej w Warszawie
  • Instytut Chemii Bioorganicznej PAN w Poznaniu
  • Instytut Chemii Fizycznej w Warszawie
  • Instytut Farmakologii PAN Kraków
  • Instytut Genetyki i Hodowli Zwierząt PAN – Jastrzębiec
  • Instytut Genetyki Roślin PAN w Poznaniu
  • Instytut Immunologii i Terapii i Doświadczalnej PAN Wrocław
  • Instytut Medycyny Doświadczalnej i Klinicznej PAN Warszawa
  • Instytut Medycyny Wsi Lublin
  • Instytut Rozrodu Zwierząt i Badań Żywności PAN w Olsztynie
  • Zakład Ichtiobiologii i Gospodarki Rybackiej PAN – Gołysz
  • Międzynarodowy Instytut Biologii Molekularnej i Komórkowej Warszawa
  • Instytut Fizjologii i Patologii Słuchu Warszawa
  • Instytut Hodowli i Aklimatyzacji Roślin /oddziały
  • Instytut Matki i Dziecka Warszawa
  • Instytut Ochrony Roślin PIB w Poznaniu
  • Instytut Włókien Naturalnych i Roślin Zielarskich Poznań
  • Instytut Psychiatrii i Neurologii Warszawa
  • Instytut Zootechniki PIB – Kraków
  • Muzeum i Instytut Zoologii PAN Warszawa
  • Narodowy Instytut Geriatrii, Reumatologii i Rehabilitacji Warszawa
  • Narodowy Instytut Leków Warszawa
  • Narodowy Instytut Zdrowia Publicznego – PZH Warszawa
  • Państwowy Instytut Weterynaryjny PIB Puławy
  • Wojskowy Instytut Higieny i Epidemiologii w Warszawie
  • Europejskie Regionalne Centrum Ekohydrologii PAN Łódź
  • Akademia Wychowania Fizycznego Poznań
  • Uniwersytet Gdański
  • Uniwersytet im. A. Mickiewicza w Poznaniu
  • Uniwersytet Jagielloński Kraków
  • Uniwersytet Jana Kochanowskiego Kielce
  • Uniwersytet im. Kardynała Stefana Wyszyńskiego Warszawa
  • Uniwersytet Kazimierza Wielkiego w Bydgoszczy
  • Uniwersytet Łódzki
  • Uniwersytet Mikołaja Kopernika w Toruniu
  • Uniwersytet Przyrodniczy Lublin
  • Uniwersytet Przyrodniczy Poznań
  • Uniwersytet Przyrodniczy Wrocław
  • Uniwersytet Przyrodniczo Humanistyczny Siedlce
  • Uniwersytet Rolniczy Kraków
  • Uniwersytet Rzeszowski
  • Uniwersytet Szczeciński
  • Uniwersytet Śląski Katowice
  • Uniwersytet Technologiczno-Przyrodniczy w Bydgoszczy
  • Uniwersytet Wrocławski
  • Uniwersytet Warszawski
  • Uniwersytet Warmińsko-Mazurski Olsztyn
  • Uniwersytet Medyczny w Białymstoku
  • Uniwersytet Medyczny Lublin
  • Uniwersytet Medyczny Łódź
  • Uniwersytet Medyczny Poznań
  • Uniwersytet Medyczny Wrocław
  • Uniwersyteckie Centrum Kliniczne WUM w Warszawie
  • Gdański Uniwersytet Medyczny
  • Pomorski Uniwersytet Medyczny Szczecin
  • Śląski Uniwersytet Medyczny w Katowicach
  • Warszawski Uniwersytet Medyczny
  • Zachodniopomorski Uniwersytet Technologiczny w Szczecinie
  • Politechnika Gdańska
  • Politechnika Łódzka
  • Politechnika Poznańska
  • Szkoła Główna Gospodarstwa Wiejskiego Warszawa
  • Wojskowa Akademia Techniczna Warszawa
  • Centrum Onkologii w Warszawie
  • Wojewódzki Inspektorat Weterynarii Opole
  • Wojewódzka Stacja Sanitarno-Epidemiologiczna Bydgoszcz
  • Wojewódzka Stacja Sanitarno-Epidemiologiczna Lublin
  • Wojewódzki Szpital Specjalistyczny we Wrocławiu
  • Zakład Ichtiobiologii i Gospodarki Rybackiej PAN w Gołyszu
  • Fundacja Bioedukacji Warszawa
  • Fundacja Uniwersytet Dzieci Kraków
  • Adamed Pharma SA (Pieńków)
  • Alab Laboratoria sp.zo.o.Warszawa
  • Alchem sp.zo.o.Poznań
  • Analityk Ewa Kowalczyk Warszawa
  • Analityk Genetyka sp.zo.o.Warszawa
  • Animal-Lab Laboratorium weterynaryjne w Łodzi
  • A-Z Medica sp.zo.o.Gdańsk
  • Bacto-Tech sp.z o.o. Toruń
  • Bioceltix sp.z o.o. Wrocław
  • Bioton SA Warszawa
  • Cellis sp.zo.o.Warszawa
  • CELON PHARMA SA
  • Centra Genetyki Medycznej Genesis sp.z o.o. Poznań
  • Celther Polska sp.z o.o. Zakroczym
  • Curiocity Diagnostics sp.zo.o.Warszawa
  • EURx sp. z o.o.
  • Genoplast Rokocin
  • Hurt-Chem Hurtownia Odczynników Chemicznych Duchnice
  • Instytut Bioinfobank Sp Z.o.o. Poznań
  • KRAKCHEMIA SA Kraków
  • Laboratorium Dermatologiczne Derma-Lab sp. z o.o. sp.k Wrocław
  • Laboratorium Badań Genetycznych sp. z o.o. Lublin
  • Medycyna Diagnostyczna PUM sp. z o. o. Szczecin
  • OncoArendi Therapeutics SA Warszawa
  • Oncogene Diagnostics sp.z o.o. Kraków
  • Personather sp.z o.o. Konstantynów Łódzki
  • Read Gene SA Szczecin
  • RDLS sp.z o.o. Warszawa
  • Rex Company Wrocław
  • RYVU-Therapeutis SA Kraków
  • Szpital Kliniczny Dzieciątka Jezus w Warszawie
  • Samodzielny Publiczny Centralny Szpital Kliniczny Warszawa
  • Samodzielny Publiczny Centralny Szpital Kliniczny Warszawa
  • Salus Medycyna sp. zo.o. Siedlce
  • Selvita SA Kraków
  • Stowarzyszenie dla Natury Wilk Godziszka
  • Stowarzyszenie Polskiej Grupy ds Leczenia Białaczek u Dorosłych Gliwice
  • SoftSystem sp.z o.o. Rzeszów
  • Synevo Polska University of Warsaw, Poland

Badania

Opis badań

Since the 1990s, the facility has been involved in many national and international sequencing projects. In 2008, we were the first laboratory in Poland to use NGS (454/Roche) in genomic research. Since 2014, we have introduced third-generation long-read sequencing, which has been used to produce highly accurate de novo assemblies of small- to medium-sized genomes in our facility. The sequencing team has participated in multiple functional genomics projects, comprising the sequencing of yeast (Yeast. 1995, 11[12]:1179-86), Paramecium (Trends Genet. 2001;17[6]:306-8.), and potato (Potato Genome Sequencing Consortium International, Nature. 2011;475[7355]:189-95) genomes and their functional analyses. Experienced staff in the sequencing unit were also involved in several microbial genomics projects that focused on Pseudomonas sp. (BMC Genomics. 2020;21[1]:1-2), Lactobacillus sp. (Appl Microbiol Biotechnol. 2019;103[18]:7617-34), Sinorhizobium sp. (J Biotechnol. 2013;164[4]:479-88), and organelles of Prototheca wickerhamii (Front Plant Sci. 2020;11:1296). We are currently dealing with sequencing the genome and transcriptome analysis of Prototheca wickerhamii, a colorless microalgae. P. wickerhamii belongs to the genus of the only known plant that has consistently been implicated in a range of clinically relevant opportunistic infections in both animals and humans. Another project that is currently being performed in our facility involves the genetic epidemiology of Streptococcus pneumoniae serotype 19A strain resistance to macrolide antibiotics, the most common bacterial cause of acute respiratory infection and otitis media.

Wybrane publikacje

  • Sequencing and Analysis of the Complete Organellar Genomes of Prototheca wickerhamii. Bakuła Z, Gromadka R, Gawor J, Siedlecki P, Pomorski JJ, Maciszewski K, Gromadka A, Karnkowska A, Jagielski T. Front Plant Sci. 2020 Sep 1;11:1296. doi: 10.3389/fpls.2020.01296. eCollection 2020
  • Bacterial community dynamics in spontaneous sourdoughs made from wheat, spelt, and rye wholemeal flour. Boreczek J, Litwinek D, Żylińska-Urban J, Izak D, Buksa K, Gawor J, Gromadka R, Bardowski JK, Kowalczyk M. Microbiologyopen. 2020 Apr;9(4):e1009. doi: 10.1002/mbo3.1009. Epub 2020 Feb 11
  • High-throughput sequencing approach in analysis of microbial communities colonizing natural gas pipelines. Staniszewska A, Kunicka-Styczyńska A, Otlewska A, Gawor J, Gromadka R, Żuchniewicz K, Ziemiński K. Microbiologyopen. 2019 Aug;8(8):e00806. doi: 10.1002/mbo3.806. Epub 2019 Feb 6. PMID: 30729757; PMCID: PMC6692550
  • Draft Genome Sequences of the Highly Halotolerant Strain Zygosaccharomyces rouxii ATCC 42981 and the Novel Allodiploid Strain Zygosaccharomyces sapae ATB301T Obtained Using the MinION Platform. Bizzarri M, Cassanelli S, Pryszcz LP, Gawor J, Gromadka R, Solieri L. Microbiol Resour Announc. 2018 Aug 2;7(4):e00874-18. doi: 10.1128/MRA.00874-18. eCollection 2018 Aug
  • Genome sequence and analysis of the tuber crop potato. Potato Genome Sequencing Consortium, Xu X, Pan S, Cheng S, Zhang B, Mu D, Ni P, Zhang G, Yang S, Li R, Wang J, Orjeda G, Guzman F, Torres M, Lozano R, Ponce O, Martinez D, De la Cruz G, Chakrabarti SK, Patil VU, Skryabin KG, Kuznetsov BB, Ravin NV, Kolganova TV, Beletsky AV, Mardanov AV, Di Genova A, Bolser DM, Martin DM, Li G, Yang Y, Kuang H, Hu Q, Xiong X, Bishop GJ, Sagredo B, Mejía N, Zagorski W, Gromadka R, Gawor J, Szczesny P, Huang S, Zhang Z, Liang C, He J, Li Y, He Y, Xu J, Zhang Y, Xie B, Du Y, Qu D, Bonierbale M, Ghislain M, Herrera Mdel R, Giuliano G, Pietrella M, Perrotta G, Facella P, O’Brien K, Feingold SE, Barreiro LE, Massa GA, Diambra L, Whitty BR, Vaillancourt B, Lin H, Massa AN, Geoffroy M, Lundback S, DellaPenna D, Buell CR, Sharma SK, Marshall DF, Waugh R, Bryan GJ, Destefanis M, Nagy I, Milbourne D, Thomson SJ, Fiers M, Jacobs JM, Nielsen KL, Sønderkær M, Iovene M, Torres GA, Jiang J, Veilleux RE, Bachem CW, de Boer J, Borm T, Kloosterman B, van Eck H, Datema E, Hekkert Bt, Goverse A, van Ham RC, Visser RG. Nature. 2011 Jul 10;475(7355):189-95. doi: 10.1038/nature10158

Nagrody i wyróżnienia

  • Anna Kowalska. AgroBioTop Prize. 2014. The Committee of Biotechnology of the Polish Academy of Sciences, Poland
  • Jan Kowalski. Fellowship for Outstanding Young Scientists. 2010. Ministry of Science and Higher Education, Poland

Publikacje