GENOME/EXOME SEQUENCING (WGS)
Whole Genome (re) Sequencing (WGS) means the (re) sequencing of a whole genome (WGS), be it Viral, Bacterial, Fungal, Animal or Plant. Provides the most complete map of an organism's genetic makeup.
The method allows the analysis of various genetic polymorphisms (single base variations, insertions, deletions, inversions, complex rearrangements, copy number variation). Unlike exome sequencing, WGS covers all coordinates of the entire genome of an organism, which is probably the most important molecular data.
For bacteria, this approach is highly effective for studying virulence, drug resistance, or novel drug targets. Likewise, comparative genomics for eukaryotes using resequencing or de novo assembly is the best approach for obtaining high-resolution genomic variations.
When a Reference is not available, the de novo assembly allows to obtain the sequence of the whole genome. For proper de novo assembly of the whole genome, a combination of shorter reads and longer contigs allows for the characterization of any genome.
Whole Exome Sequencing (WES) means the selective sequencing of the coding region (Exons), which constitutes for example 1-2% of the human genome. It is a cheaper and more effective way of sequencing than whole genome sequencing. Exome sequencing can be effectively used in research for rare diseases, cancer genomics, and genetic disorders.
Bio-Fab Lab uses different methods of exon capture to provide the exome sequencing service.
TRANSCRIPTOME SEQUENCING (RNA-Seq)
RNA-Seq (RNA sequencing), also called Whole Transcriptome Sequencing (WTS), uses third generation sequencing to detect presence and the quantity of RNA in a biological sample at a given specific time or under certain physiological, pathological or experimental conditions.
Specifically, RNA-Seq allows examining isoforms, post-transcriptional modifications, gene fusions, mutations / SNPs and variations in gene expression over time or differences in gene expression in different groups or treatments. In addition to mRNA transcripts, RNA-Seq can examine different populations of RNA including total RNA, small RNAs, such as miRNAs, tRNAs, and Ribosom profiling (Ribo-seq). RNA-Seq can also be used to determine exon / intron boundaries and verify or modify the 5 'and 3' limits of previously annotated genes.
Whole transcriptome sequencing is an important advance in the study of gene expression over traditional microarray-based approaches as it provides a comprehensive view of a cellular transcriptional profile at a given biological time allowing for not only qualitative but also quantitative analysis of the transcriptome. It also provides information on different variations that occur at the RNA level, of post-transcriptional modifications such as junction variants and isoforms. The information that can be obtained is dependent on the Coverage (see Bioinformatics section)
Expression profile analysis, from RNA-Seq sequencing data, offers a more complete view of the transcription level of the individual RNAs under study.
Current standards and guidelines for RNA-seq require that the average library insert size be 200 base pairs. Experiments should have two or more replicates. Each replicate is expected to have 30 million aligned reads, even though early designs aimed for 20 million aligned reads.
ASSAY for TRASPOSASE-ACCESSIBLE CHROMATIN (ATAC-Seq)
The transposase accessible chromatin experiment followed by sequencing (ATAC-Seq) provides the genomic profiles of chromatin accessibility. Briefly, the ATAC-Seq method works as follows: the loaded transposase inserts the sequencing primers into open chromatin sites of the genome and the reads are then sequenced. The ends of the reads indicate the open chromatin sites.
SMALL RNA SEQUENCING
Small noncoding RNA-seq is an RNA-Seq protocol used to sequence different species of small RNAs such as microRNAs (miRNAs). miRNA-Seq differs from other forms of RNA-seq in that the input material is often enriched for small RNAs, 17 to 35 nucleotides. miRNA-Seq allows to examine tissue-specific expression patterns, disease associations, miRNA isoforms and to discover previously uncharacterized miRNAs. MiRNA dysregulation plays an important role in numerous diseases, making miRNA-seq an important tool for diagnostics and prognostics, as biomarkers and / or disease predictors.