“SHERLOCK is exciting because it represents a sensitive approach to detect mutations, much like PCR, but it does not require a thermocycler and could work as a bedside test.” - Dr Tilmann Buerckstuemmer, Global Head of Innovation at Horizon Discovery
We all know how vital quality control is for our samples. A lot has gone in to developing useful QC metrics for genomics experiments – primarily due to their high cost. Skipping this step will waste both time and money.
There are 3 main areas where QC can be applied to NGS:
- On the starting nucleic acids
- After Library preparation
"RNA-Seq is the most likely NGS technique to be used by researchers in 2017 due to its versatility"
RNA-seq has become a ubiquitous tool in both biological and medical research. Much of the RNA-seq analysis done is still for differential gene expression from poly-adenylated mRNAs; but the success of RNA-seq can also be seen in the rapid increase in knowledge about biological systems and the large number of distinct variants of the method. The combination of these factors allow us to go much further than the 'simple' 3’ gene expression microarrays and now opens up the possiblities of: splicing analysis, differential allele expression, variant detection, alternative start/stop, gene fusion detection, RNA editing and eQTL mapping to name but a few...
Do you remember when…?
Do you remember when Frederick Sanger and his colleagues invented dideoxynucleotide chain termination sequencing back in 1977? Technology has come on a long way, and the recent advancements now means that we're doing more sequencing now than ever before.
Next Generation Sequencing
Next Generation Sequencing (NGS), has become a universal tool in diverse industries; most recently moving into the clinic for patient diagnosis. However, if labs wish to analyze patient-derived materials they must first face a hurdles labs to optimize and validate their workflow including determining the exact test limitations.
It is due to the unequivical interest and increasing popularity of NGS based tumor testing that specific guidelines for detecting tumor variants have been established, including by the State of New York Health Department1 advizing on how to determine an assay’s limit of detection to name but one parameter. Based on the recommendations, they state that samples should have enough sequence data for a minimum average coverage of 500x so that minor allele frequencies of 5% can be reliably detected. However, with newer technologies bestoying increasing importance on low allelic frequencies and coverage bias issues (more below about this), should we be aspiring to push this limit of detection even further?
Droplet Digital™ PCR, labelled the third generation of PCR technology, enables the absolute quantitation of target DNA. The platform allows for the enrichment of rare target sequences, the detection of small differences in target concentration and the determination of copy number without the need for a standard curve.
In this study we show the Bio-Rad QX100™ ddPCR™ platform was able to detect mutant alleles (BRAF V600E, BRAF V600K, KRAS G12D, KRAS G12V, EGFR ΔE746-A750) present at frequencies as low as 0.05%. In addition the platform enabled the detection of mutant alleles present as low as 3.5% using DNA extracted from the FFPE Reference Standards.
To read the complete application note, including assay optimisation, validation of Copy Number assays and estimated costs of detecting different allele frequencies on this platform download the complete application note.
Isolation of genomic DNA from formalin fixed paraffin embedded (FFPE) tissues is a critical step for molecular diagnostic (MDx) assays. It is essential that the maximum amount of DNA is recovered from the FFPE tissues and its quality is sufficient to perform the necessary MDx assays (e.g. QPCR, Sanger Sequencing, Pyrosequencing™).
To investigate which DNA extraction kit was most appropriate for recovering the optimum DNA yield and concentration from low cell numbers, Horizon generated a custom FFPE cell line block, and performed extraction of DNA from FFPE using five comparable methods:
- Promega Maxwell® 16 FFPE Tissue LEV DNA Purification Kit
- Promega MagneSil® Genomic, Fixed Tissue System Kit
- Promega ReliaPrep™ FFPE gDNA Miniprep System
- Qiagen DNeasy Blood & Tissue Kit
- Roche Cobas® Sample Preparation Kit
The high-throughput and increasingly affordable nature of Next-Generation Sequencing (NGS) has led to its expanded use in routine clinical procedures. The relative simplicity of targeted enrichment cancer panels (available from a number of commercial providers) allows routine laboratories to simultaneously analyze the coding (exonic) regions of multiple cancer-related key genes. Combine this with the statistic diagnostic testing now influencing over 70 percent of all health care decisions1, the setting up or transitioning to NGS-based oncology panels for labs has never been more important.