Pracownie usługowo-badawcze
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Dr Robert Gromadka
Środowiskowa Pracownia Sekwencjonowania i Syntezy DNAZakres 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
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 (z afiliacją IBB PAN)
Zespół
- Robert Gromadka, PhD, Kierownik Pracowni, ORCID: 0000-0002-9941-6692
- Beata Babińska, Pracownik
- Jarosław Cieśla, PhD, Pracownik, ORCID: 0000-0003-0695-7201
- Małgorzata Filipiak, Pracownik
- Jan Gawor, Pracownik, ORCID: 0000-0003-3800-2557
- Ewa Kalińska, Pracownik, ORCID: 0000-0003-0911-2677
- Karolina Wylot, Pracownik
- Karolina Żuchniewicz, Pracownik, ORCID: 0000-0002-7097-4025