NGS SEQUENCING NEW TECHNOLOGY
Technology – DNA sequencing has redefined research in biological sciences. Next generation sequencing technology sequences millions of DNA fragments in a parallel sequencing, which provides unprecedented scalability for projects of all sizes, including whole genome sequencing, exome sequencing, RNA-seq etc. The potential sensitivity is nearly limitless, correlating with the customizable depth of coverage, which permits the detection of very rare mutations, transcripts, and biomarkers.
HiSeq sequencer is an advanced sequencing method that uses Illumina technology. This enables to receive up to 600 GB (600x nucleotides) in 6 trillion paired end reads during 11 days. It consists of 16 lines on which sequencing is performed separately. For each line, 170 million of PE reads is generated.
MiSeq sequencer employs sequencing-by-synthesis Illumina technology, which allows to obtain up to 15 GB (15×109 bases) in 45 million paired reads from one sequencing trial. MiSeq is the only desktop sequencer that can produce 2 x 300 paired-end reads in a single run. This allows small genome sequencing and assembly, and enables detection of target variants with unmatched accuracy, especially within homopolymer regions. Now, even more samples can be processed in less time while generating more reads per run than any previous versions. All of this can be achieved using the targeted gene and small genome sequencer with the shortest sample-to-data workflow.
The SBS technology supports both, single read and paired-end read libraries with read lengths up to 150bp for HiSeq, and up to 600bp (2x300bp) with the newest MiSeq chemistry.
Genomic Laboratory offers comprehensive NGS sequencing services, starting from material isolation (tissue, leukocyte pellet, blood, swab, etc.), purification, fluorescence quantification (Qubit), quality assessment (Bioanalyzer), library preparation, sequencing and bioinformatics analysis.
Genomic Laboratory of the DNA Research Center can sequence your samples, starting from any kind of material, for specific applications (read more below), but you also have the possibility to sequence libraries you have prepared yourself. You can buy sequencing on the HiSeq per lane, or for the whole flow cell.
If you are interested in any other application or want to discuss your project possibilities.
CURRENTLY AVAILABLE APPLICATIONS:
· Genome analysis
– whole genome sequencing
– de novo sequencing of large and small genomes (bacteria, yeast, viruses)
– Mate Pair sequencing,
– exome sequencing,
– targeted resequencing,
– plasmid sequencing,
– amplicon sequencing,
· Transcriptome analysis
– small RNA sequencing
· Epigenome analysis
– Genome Chromatin IP (ChIP) Sequencing
– methylation analysis
· Additional services
– expression microarrays
– metagenomics (16S rRNA)
– genetic predisposition panels:
· Cancer panels o germline mutations (genetic predispositions) – entire spectrum or specific cancer panels:
· Panel 170 PLUS includes genes associated with both common (e.g., breast cancer, prostate, hematologic malignancies, colorectal, lung, kidney) and rare cancers. Targets over 1700 exons, spanning 94 genes of interests and additional 284 SNPs occurring in 78 different genes associated with predisposition towards cancer.
· Panel “For Him” includes genes associated with the most common male cancers, like: prostate cancer, colorectal cancer, lung cancer, testicular germ cell tumor. Targets exons spanning 54 genes of interests and additional 45 SNPs in 45 different genes associated with predisposition towards the most common male cancers.
· Panel “For Her” includes genes associated with the most common female cancers, like: breast, colorectal, lung and ovarian cancer. Panel “For Her” targets exons spanning 48 genes of interests and additional 22 SNPs in 22 different genes associated with predisposition towards the most common female cancers.
· Somatic mutations – The test detects mutations across 26 genes (targeted with 174 amplicons) in cancer cells, mutations which may be involved in progression of cancerous tissue in patients with solid tumors. Many of these genes, including KRAS, EGFR and BRAF, have been associated with cancers such as melanoma, colorectal, ovarian and lung. With “somatic cancer panel”, Genomic Laboratory is able to reliably detect mutations occurring with frequency even below 5%.
· Cardiomyopathy panel – targets 46 genes linked to inherited cardiomyopathy. This Panel targets genes linked to inherited cardiomyopathies, including Hypertrophic Cardiomyopathy (HCM), Dilated Cardiomyopathy (DCM), Restrictive Cardiomyopathy (RCM), Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC), Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT), and Left Ventricular Non-Compaction (LVNC). Additional content covers several syndromes, including Danon and Fabry disease, Barth syndrome, and Transthyretin Amyloidosis — all of which can be present with isolated cardiomyopathy.
· Autism panel – targets over 1 700 exons, spanning 101 genes linked specifically to autism. These are genes reported in the Online Mendelian Inheritance in Man (OMIM) database for autism research; genes with recurrent mutations resulting in developmental disability; genes with reported mutations found in case of studies involving developmental disability characteristics; and combined autism-relevant genes (e.g., AutismKB). Genes neighboring strong association signals were excluded in the absence of published reports of mutations.
· Inherited disease panel – targets over 8 800 exons, spanning 552 genes of interest linked specifically to a wide spectrum of inherited diseases. It includes coding exons, intron-exon boundaries and regions known to harbor pathogenic mutations.
Price-list for MiSeq experiments:
200 – 500 PLN net – one library preparation for DNA sequencing
900 – 1000 PLN net- one library preparation for RNA and small RNA sequencing
9 000 – 12 000 PLN net – sequencing on MiSeq
Price-list for HiSeq experiments:
400 – 500 PLN net – one library preparation for DNA sequencing
800 – 1000 PLN net – one library preparation for RNA and small RNA sequencing
13 000 – 15 000 PLN net – price for one line on HiSeq (no matter how many samples to be sequenced).
Prices may vary depending on the number of samples and complexity of experiment. Please contact us for the specific pricing for your project.
Beyond producing high-quality sequencing data on our various NGS platforms, DNA Research Center offers you the following additional services:
· DNA/RNA isolation
· Material quantification (fluorescent method – Qubit)
· Material quality analysis (Experion)
· Library preparation
· PCR and qPCR services
· Sanger DNA sequencing
Service step by step:
1 – assistance in the experimental set-up
2 – library preparation
3 – NGS sequencing on MiSeq or HiSeq
4 – bioinformatics analysis
Please define the preferred sample pooling method (= how many samples are to be sequenced in 1 line?) – depending on expected coverage. You can also contact us and ask for assistance.
E-mail address for NGS services: Błąd! Nieprawidłowy odsyłacz typu hiperłącze.
GENERAL DESCRIPTION OF APPLICATIONS
De novo whole genome sequencing
De novo sequencing is crucial for better understanding of organisms functioning. It allows us to explore the whole genome without the need to use any reference sequences. De novo genome sequencing can be carried out for each organism, a plasmid, an artificial chromosome, etc.
Whole genome and amplicons resequencing
Resequencing of the whole genome or amplicons is used to obtain information about the genetic variations with respect to the reference sequence. This technique allows us to obtain information about the new variants, variants stored in the databases (mainly in coding regions), variants located in non-coding regions which are not detectable by array methods or exome sequencing. An important advantage of the MiSeq platform is the ability to sequence amplicons. In contrast to the traditional Sanger sequencing methods, which gives maximum of 2-fold coverage (coverage), NGS sequencing allows to obtain even a few thousand–fold coverage depending on the complexity and needs of the project.
Exome and target sequencing
Exome sequencing (targeted exome capture) is an efficient strategy to selectively sequence coding regions of the genome as cheaper and still effective alternative to whole genome sequencing. Exome sequencing involves sequencing of protein coding regions and around 200 surrounding nucleotides. Exome sequencing is particularly important in the analysis of rare mutations associated with genetic disorders that can be reliably interpreted using a variety of publicly available genetic databases (e.g. dbSNP).
Metagenomics: Sequencing of hipervariable regions of 16S RNA gene
16S ribosomal RNA (rRNA) sequencing is a common amplicon sequencing method used to identify and compare bacteria present within a given sample. 16S rRNA gene sequencing is a well-established method for studying phylogeny and taxonomy of samples from complex microbiomes or environments that are difficult or impossible to study. Data from 16S studies are used to improve the sensitivity and specificity of taxonomic assignments, down to the species level.
Unlike capillary sequencing or PCR-based approaches, next-generation sequencing (NGS) is culture-free method that enables analysis of the entire microbial community within a sample, including identification of species that may not be found using other methods. With the ability to combine many samples in a sequencing run, microbiology researchers can use NGS-based 16S rRNA sequencing as a cost-effective technique to identify strains that may not be found using other methods.
Thanks to its speed, efficiency and accuracy, NGS sequencers offer a new option for the classic RNA sequencing and transcriptome analysis. Transcriptome is the full set of transcripts, comprising mainly mRNA, but also small RNA molecules such as tRNA and rRNA. RNA-seq method is an alternative for expression microarray. Analysis can be performed for every species for which there is a reference genome. Furthermore, expression data received from the NGS sequencer can be analyzed using many of the tools commonly used in the microarray analysis, in the hierarchical clustering.
RNA sequencing (RNA-Seq) has revolutionized exploration of gene expression. Advances in RNA sequencing workflow, from sample preparation through data analysis, enable rapid profiling and deep investigation of transcriptome.
Next-generation RNA sequencing enables researchers to:
· Identify and quantify both rare and common transcripts
· Align sequencing reads across splice junctions, detect isoforms, novel transcripts and gene fusions
· Derive strand information with high precision
· Perform robust whole-transcriptome analysis on a wide range of samples, including low-quality samples
SMALL RNA SEQUENCING
Small RNA sequencing is the most reliable method for the identification and profiling of small RNAs which include microRNA, siRNA, Pirn and rRNA. MicroRNA play a key role in the growth, development, differentiation and cell death. Therefore, is an interesting research topic throughout the world. Moreover, miRNA profiles are increasingly regarded as biomarkers, in particular in the field of cancer research, which suggests that may play a role as a new class of oncogenes or tumor suppressors. Small RNA sequencing is becoming more and more popular technique that enables development of new drugs and molecular biomarkers.
ChIP-Seq is a powerful tool for genome-wide mapping of histone modifications, protein-DNA interactions, and identifying consensus protein-binding sites in DNA. By combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing, ChIP-Seq can be used to accurately survey interactions between protein, DNA, and RNA, enabling the interpretation of regulation events central to many biological processes and disease states.
Leveraging Illumina’s industry-leading sequencing technology, ChIP-Seq can identify a broad range of protein/nucleic acid interactions with confidence, generating millions of counts across multiple, indexed samples per lane for cost-effective and precise analysis.
WGSB – The Whole Genome Sequencing after reaction with bisulfite is an effective technique for DNA methylation research. WGBS combines bisulfite treatment of DNA with high throughput sequencing which can generate high accuracy methylomes and enables performing error-free genomic scale analysis. WGBS technology may be used in epigenetic studies, including embryonic development, cell differentiation and similarity, transubstantiation of stem and adult cells, and cell response to environmental signals (hormones, nutrients, stress, damage).
• Accurate analysis of methylation patterns for each cytosine
• Generates a complete methylome map for each organism
• Gold Standard in the analysis of DNA methylation
We also offer targeted bisulfite sequencing which allows researchers to receive significant data sets for regions of interest from a large number of samples while avoiding the expense and time required for genome-wide sequencing. This is particularly well suited for the validation of putative biomarker candidates.