Genomics and Epigenetics
Next Gen Sequencing or Microarray: How to Make the Choice
You’re about to start that big project you’ve been dreaming of for years. You’ve identified a potential miracle compound and want to figure out how it affects gene expression. But how are you going to do it: with next gen sequencing or a microarray? Especially if you are new to this area of research, the…
Read MoreDon’t Get Lost in RNA-seq Translation: RNA Sequencing the NGS Way
DNA sequencing (PCR, Sanger or next-generation sequencing (NGS)) is a now familiar part of any molecular biology lab. But ‘RNA-seq’, the so-called “Cinderella of genetics”, is now becoming the belle of the ball, providing new insights into this most central molecule of the ‘central dogma’. The many flavors of RNA Whilst genomic DNA is the…
Read MoreHow Bisulfite Pyrosequencing Works
Bisulfite pyrosequencing is becoming a routine technique in molecular biology labs as a method to precisely measure DNA methylation levels right down to the single base. The technique allows for detailed and high resolution analysis of DNA methylation at specific genomic regions. How to detect the 5th base? Methylation of any of the four nucleotides…
Read MoreTower of Babel: Next Generation Sequencing Provides New Insights on Chromosome Construction
Biologists have long appreciated the complexity of genome organization, but until recently lacked the tools to discern the intricacies of this puzzle. Now, thanks to some handy cross-linking, careful amplification, and (of course!) next generation sequencing, teams from Massachusetts are taking us down the rabbit hole, with some surprising findings from Wonderland. Bend Over Backwards…
Read MoreThe Irish Potato Famine: NGS Unearths The Fungus Responsible For Over 1 Million Deaths
The Irish Famine (or ‘Great Potato Famine’ if you live outside the Emerald Isle) killed one million people and forced another million to leave the country between 1845 and 1852. It was caused by a blight on the country’s main food stock- the Irish ‘Lumper’ potato. Now, researchers have identified the genome of the blight…
Read MoreConrad Waddington and his epigenetic landscape
I was first introduced to Conrad Waddington’s epigenetic landscape when reading ‘The epigenetic revolution’, a fantastic introduction to epigenetics, and in my opinion, a must read for anyone who is looking for an entertaining and enjoyable introduction to this fascinating field. In his model, Waddington likens the process of cellular differentiation to a marble, which…
Read MoreGetting the most out of your human DNA methylation studies
The field of epigenetics is exploding and given the strong links between epigenetic state and disease, the need to study markers like DNA methylation in humans is very relevant. This article outlines some of the main factors you should be taking into account in your study of DNA methylation in human tissues. Here goes: Biological…
Read MoreWhat To Do When Whole Genome Sequencing Yields Unexpected Results: Is There A One-Size-Fits-All Answer?
Whole genome sequencing (WGS) is becoming increasingly common. Doctors now routinely order it for patients with puzzling diseases. The NHS (National Health Service in the UK) has declared that it will sequence 100,000 genomes over the next few years. Increase WGS…increase ethical questions The direct-to-consumer company 23andme has been experimenting with whole exome sequencing (WES), and another company, DNA…
Read MoreGalaxy: A Free NGS Workflow Management System
Most ‘wet lab’ biologists do not have much computer programming experience, which can make downstream analysis of next generation sequencing results a bit daunting. After the sequencing platform spits out your data, what do you do with it? That’s where Galaxy comes in. What is Galaxy? Galaxy is a bioinformatics workflow management system, created by collaboration…
Read MoreShearing DNA For Next Generation Sequencing: Which Method Should I Choose?
Next-generation sequencing (NGS) really has taken the world by storm! In NGS, millions of short ‘read’s are sequenced in a short space of time, leaving you with vast amounts of data to analyze! For all NGS platforms, the input sample (i.e. your cell free DNA) must be cleaved into short sections or fragments prior to…
Read MoreSolid Phase Reversible Immobilization: How To Get A Bead On The Clean-up Of Your NGS Libraries
“Any sufficiently advanced technology is indistinguishable from magic.” – Arthur C. Clarke In the fast-moving field of next generation sequencing, standard practices are evolving rapidly. Today, more and more labs are using Solid Phase Reversible Immobilization (SPRI) beads instead of gel purification in the preparation of libraries for sequencing. A crucial step, not for the…
Read MoreHow To Troubleshoot Your DNA Libraries For 454 Next Generation Sequencing- An Essential Guide.
For those of you who prepare your own DNA libraries, this article will cover the most critical aspects of library preparation to ensure a successful sequencing run. Previous Bite Size Bio articles have covered the basics of how 454 sequencing works, so give those a quick review if you are unfamiliar with the process. This video is also highly…
Read MoreWhy Is It Important To Run Your NGS Gels Consistently?
Size Selection via Gel Electrophoresis Whether you are using NGS for whole genome sequencing, SNP variant analysis, HLA typing, HLA matching, or even transcriptome or miRNA analysis by RNA-seq, size selection is an extremely important consideration for optimum results. Precise size selection can increase sequencing efficiency, save money and improve genome assemblies, as well as…
Read MoreWhere Did It All Go Wrong?! Quality Control For Your NGS Data
You’ve carefully collected your samples, extracted nucleic acids and made your first set of next-generation sequencing libraries. How are you going to know if the data you get back is any good and whether it will be worth the effort in learning how to do the analysis? Who is to blame? Fortunately, there are several…
Read MoreIgnore The Ticking Bomb At Your Peril: RNA-Seq Normalization- A Lurking Problem And It’s Solution.
You have spent days, if not weeks, at the bench setting up the treatment and control samples for that crucial experiment. You submitted your cDNA library for sequencing and after a few weeks of waiting anxiously you get back a list of differentially expressed genes. Hooray?! Hold on- not quite yet! There is something you…
Read MoreA Beginners’ Guide to Non-coding Sequence Alignment
There is no such thing as “junk” DNA Until recently, vast areas of the genome had been denounced as “junk” DNA, because they do not encode proteins. However, it has become clear that these regions have a large diversity of other functions, from transcriptional and translational regulation to the protection of genes and genome integrity.…
Read MoreExploring The Frontiers: An Introduction to Metagenomics
Why Metagenomics? The microorganisms that we know and understand today are the ones which either cause human diseases, or are beneficial to human society in some way. From wine and cheese in food industry to the pharmaceutical industry- they are an indispensable part of our lives. Despite making good progress in understanding the microbial world,…
Read MoreHow To Identify Conserved Elements In Genes
Conserved elements are stretches of DNA sequence that are under purifying selection. That means mutations leading to a change of function in this part of the DNA are detrimental to the organism and will not become fixed in the genome, but rather discarded by natural selection. The level of conservation between species gives an idea…
Read More7 Tips for Preparing Chromatin for ChIP from Tissues (Rather than Cells)
A commonly used technique in epigenetics is Chromatin Immunoprecipitation, or ChIP for short. This technique can show you whether a certain protein (e.g. transcription factor or histone modification) binds to DNA, when in its native conformation, namely chromatin. Insightful, but difficult This information can be very insightful, but difficult to obtain. Most protocols and suggestions…
Read More10 Ways to Improve Your Bisulfite Sequencing Results
The importance of epigenetics in biology is increasingly acknowledged (if you’re not convinced yet, read my crash course). One commonly studied epigenetic mark is CpG methylation: cytosines that are directly followed by a guanine nucleotide (indicated by CpG), can be methylated, unlike non-CpG Cs. Since attachment of a methyl group to a cytosine can affect…
Read MoreSome Sanger Sequencing Tips and Tricks
Sanger sequencing is still a workhorse of most molecular biology labs. Even with the advent of next-generation sequencing we still need to sequence our clones and PCR products. In this article I have listed some of the tips and tricks we used in our Sanger services. (1)Dilution of BigDye: I’d expect this to be a…
Read MorePyromania: An Intro to Pyrosequencing
Anyone who is involved in DNA sequencing in one form or fashion knows there are multiple ways to skin a cat: Sanger-based, next generation (NGS), and of course the new ion torrent sequencing technology. Which technology you use is usually dependent on the questions you’re trying to answer – and how fat your wallet is.…
Read MoreBioinformatics for NGS: Open Source or Proprietary?
As many who have worked with sequence data can tell you, the biggest bottleneck in publishing papers based on sequence data is the analysis step. Most researchers are faced with a dilemma when they receive sequence data for the first time: “do I try to use these free open source programs or do I shell…
Read MoreNGS Quality Control in RNA Sequencing- Some Free Tools
RNA sequencing (‘RNA-seq’) has become one of the most widely used applications for Next-Generation Sequencing. RNA-seq can provide gene expression data more cheaply than microarray, at greater sensitivity, and without the biases inherent in an assay based on quantifying nucleic acid hybridization. RNA-seq can also provide data about alternative splicing, allele-specific expression, expression of non-annotated…
Read MoreNGS in clinical diagnostics
Thirty-five years ago, Dr. Janet Davison Rowley sat at a microscope in her lab at the University of Chicago and made a remarkable discovery in cancer biology, that leukemia is caused by the translocation of a chromosome. In other words, it is a disease of the DNA. Today, thanks to next generation sequencing (NGS), we can zoom in…
Read MoreGarbage in, Garbage out? Quality Control of Your NGS Data
So, you’ve just received a call from the core facility that you hired to prepare and sequence your libraries. The facility director tells you that the sequence data from your next generation sequencing (NGS) experiment does not look good. You panic and, perhaps, let loose a scream of frustration—aaarrrrggghhhh! This project was going to be…
Read MoreA Short History of Sequencing Part 2: the first of the next.
The Human Genome Project was successful, but hard work. The major improvements to the technology were the increases in parallelization and automation. In 2003, just as the HGP completion papers were published in Nature and Science, ABI launched the‘3730XL’. It could run 24 96-well plates per day and generate around 2 MB of sequence. Some…
Read MoreA Short History of Sequencing Part 1: from the first proteins to the Human Genome
It all started with proteins The earliest methods for sequencing were developed for proteins. In 1950, Pehr Edman published a paper demonstrating a label-cleavage method for protein sequencing which was later termed “Edman degradation”. Around the same time Fred Sanger was developing his own labelling and separation method which led to the sequencing of insulin.…
Read MoreSequencing genomes from Neanderthals to James Watson: The Roche 454 Genome Sequencer Explained
A revolution in 2005 The start of the NGS revolution was clearly marked in 2005 by the publication of the complete genome sequences of two bacterium (Mycoplasma genitalium and Streptococcus pneumonia) by 454 Life Sciences Corporation in one run of their Genome Sequencer with a 96% coverage at 99.96 % accuracy (Margulies et al. 2005).…
Read MoreHow the Ion Torrent Sequencer works
Just before Life Technologies purchased the start-up company Ion Torrent, the fledgling company was dealing with a torrent of another kind—worldwide media interest in its new sequencing technology, which promised to bring the price of next-generation, massively parallel sequencing down to $1,000 per run. Since that dramatic announcement in the summer of 2011, Life Technologies…
Read MoreSequencing-by-Synthesis: Explaining the Illumina Sequencing Technology
The “sequencing-by-synthesis” technology now used by Illumina was originally developed by Shankar Balasubramanian and David Klenerman at the University of Cambridge. They founded the company Solexa in 1998 to commercialize their sequencing method. Illumina went on to purchase Solexa in 2007 and has built upon, and rapidly improved the original technology. Millions of reactions and…
Read MoreNext Generation Sequencing: The Three Main Technologies
A paradigm shift by the Big Three As we learned last week, the Human Genome Project was accomplished using the improved Sanger method and technology from Applied Biosystems (ABI). Despite the significant technical improvements to this ‘first-generation’ technology, sequencing multiple human genomes was never going to be easy without a paradigm shift. Over the last…
Read MoreNext Generation Sequencing Channel, A Revolution in Technology
Next Generation DNA Sequencing (NGS) is a revolutionary new technology that provides biologists and medical scientists with the ability to collect massive amounts of DNA sequence data both rapidly and cheaply. This technology is having a huge impact on many aspects of biology and medicine because it can be applied in so many different ways.…
Read MoreA Crash Course in Epigenetics Part 4: Disease mechanisms and therapeutic targets
After having discussed what epigenetic mechanisms are and how we’ve learnt about what they do, it is now time to look into how epigenetics affect our lives if things do not go the way they are supposed to go. I hope I have convinced you that epigenetic processes are vital for an organism, in development…
Read MoreA Crash Course in Epigenetics Part 3: Regulated regulation
Epigenetics is the most rapidly expanding field in biology. In the second article in this series, I discussed which experimental techniques have been crucial in gaining insight into epigenetic processes. I will now shed light on what those and other methods have taught us. As described in the first article, it has been long understood…
Read MoreA Crash Course in Epigenetics Part 2: The toolbox of the epigeneticist
In the past decade, important advances have been made in the field of epigenetics. Obviously, unraveling epigenetic mechanisms has been greatly facilitated by technological developments. I’ll try to give you an impression of the types of experiments that have helped fuel those new and exciting insights. Yevgeniy Grigoryev has recently written an article on DNA…
Read MoreA Crash Course in Epigenetics Part 1: An intro to epigenetics
These days, epigenetics is a fast moving field. I don’t remember having learnt about it during my biomedical studies, some 10 years ago. Nowadays, there seems to be no way around it when studying health and disease. Increasing interest combined with recent technological breakthroughs have led to quickly expanding knowledge of its abundant and important…
Read MoreWhat Has Methylation Done For You Lately?
Epigenetics is the study of heritable changes in the phenotype of a cell or an organism that are not encoded by the genome (hence epi which means ‘above’ in Greek, and genetikos which means ‘origin’). In this article, we’ll discuss DNA methylation, a common epigenetic modification: what it is, how to detect it, and how…
Read MoreAn Introduction To ChIP-seq
ChIP-seq is a wonderful technique that allows us to interrogate the physical binding interactions between protein and DNA using next-generation sequencing. In this article, I’ll give a brief review of ChIP and introduce the chromatin immunoprecipitation sequencing technique (ChIP-seq), which combines ChIP with next-generation sequencing. What is chromatin immunoprecipitation? Chromatin immunoprecipitation (ChIP) allows us to…
Read MoreHow DNA Microarrays are Built
Previously I introduced the DNA microarray technology and described the principle behind it: hybridization between the nucleic acid sequence from the biological samples being examined and a synthetic probe immobilized and spatially arrayed on a solid surface, the microarray. In this article, I will explain how these probes are designed and positioned on the array. I…
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