Ion Torrent PGM sequencing:
The Ion Torrent sequencing platform from Life Technologies employs First PostLight™ sequencing technology (semiconductor technology). The detection of a specific nucleotide on a growing DNA strand occurs inside a well of an Ion Torrent semiconductor chip. The incorporation of the correct base triggers a chemical reaction that releases a hydrogen ion as byproduct, which in turn changes the pH of the solution. The Ion sensor captures voltage change measurements from the direct release of hydrogen ions following DNA polymerization and incorporation events get recorded as digital signals 1 and 0. This technology differs from other sequencing technologies in that no modified nucleotides, no lasers, no optics, no camera, no fluorescence, no enzyme cascade are used.
Illumina library preparation, barcoding, and sequencing:
This service includes DNA/RNA library preparation for Illumina sequencing platform for a variety of starting materials and sample barcoding to allow sequencing of multiple samples per lane. Library preparation workflow includes fragmentation, end repair and 3’ dA addition, adapter ligation, size selection and enrichment. During the enrichment PCR DNA can be barcoded for multiplexing using 8 bp indexes unique to each sample following 5’ or 3’ barcoding strategy. The barcodes can be added between the downstream bPCR adaptor and the core sequencing library adaptor.
Illumina RNA sequencing:
The service includes total RNA, small RNA or mRNA sequencing for the analysis of gene expression changes, detection of novel transcripts, small RNA discovery, alternative splice sites and cSNPs. Illumina is the only platform that offers a short-insert paired-end capability for high-resolution genome sequencing as well as long-insert paired-end reads especially important for RNA sequencing.
The method is based on the conserved 16S rDNA gene present in all bacteria (a comprehensive list of 16S rDNA sequences from gut bacteria have been deposited and categorized in the Ribosomal Database Project II ). By calculating the number of conserved 16S rDNA genes, qPCR can indirectly indicate the number of bacteria present in a given sample. Additionally, appropriate design of qPCR oligonucleotide probes (primers) that can identify the sequence of the 16S gene variable regions allows for the amplification of genes from a specific bacterium of interest whilst excluding all other bacteria, making qPCR a useful tool for quantifying specific bacteria in a complex microbial community.
The BioMark™ Systems paired with the Fluidigm Dynamic Arrays are an efficient solution for large-scale, real-time qPCR. The nanofluidic chips contain fluidic networks that automatically combine sets of samples with sets of assays. This innovative solution for real-time qPCR provides reaction densities far beyond what is possible with microtiter plates and significantly reduces the number of liquid-handling steps and the volume per reaction.
This technology expands the application boundaries of traditional real-time PCR. In contrast to real-time PCR this platform enables absolute quantification without the use of a standard curve and reference sample allowing a detection of individual DNA molecules with higher accuracy for a variety of applications.
The chip used in this system contains 20,000 individual wells and works by partitioning a standard PCR reaction into thousands of individual PCR reactions. A portion of these partitions contains the target molecule, while other partitions do not, leading to positive and negative reactions. Following amplification, the fraction of negative reactions is used to quantify the number of target molecules in the sample, all without reference to standards or controls.
We offer digital PCR service using QuantStudio 3D™ Digital PCR system from ThermoFisher Scientific. This is a chip based digital PCR platform. In contrast with droplet digital PCR the reaction is taking place in a well of the chip and not in a droplet, with fewer pipetting steps, which decreases the likelihood of cross-contamination.
- Absolute quantification of DNA molecules
- Pathogen detection and load determination
- GMO detection and contamination assessment
- Library quantification for next-generation sequencing
- Characterization of low-fold changes in mRNA and miRNA expression
- Rare cancer mutation quantification
- CNV detection
- Digital PCR does not rely on Ct values to quantify copy number, thus, PCR efficiency differences among biological samples are much less likely to affect quantitative results.
- Digital PCR does not rely on standard curve for absolute quantification.
- Digital PCR is much less sensitive to PCR inhibitors present in crude samples.
In order to confirm bacterial identity, PCR-amplified 16S rDNA fragments can be cloned into plasmids and transformed into an appropriate host (usually E. coli). Each transformed E. coli colony will contain only one 16S rDNA PCR-originated fragment from the original sample. A number of colonies are picked randomly and the insert is sequenced. The DNA sequence is then compared to a database (for example RDP II) for identification by DNA homology.
Consulting and Research Support
- Sample genomic DNA isolation
- Plasmid and virus nucleic acids isolation
- RNA isolation
- Strain typing
- Optimization of bacterial culture conditions
- High-throughput liquid handling (PCR reaction set up, sample pooling, picogreen quantification of nucleic acids)