Genomics and Epigenetics
Using dbSNP and ClinVar to Classify Gene Variants
As we discussed previously, the gaps in our understanding of the human genome make variant classification an extremely difficult job. However, with each passing day our knowledge increases, and the tools to help us become increasingly more efficient. Let’s pick up where we left off in our first article about variants. After checking Ensemble to…
Read MoreChIP-seq Workflows Run Best with Automated DNA Size Selection
Chromatin immunoprecipitation sequencing, better known as ChIP-seq, is a massively parallel approach for understanding the interactions between proteins and DNA. This is especially important for determining the activity of transcription factors, which is why it’s frequently used to learn about the complicated series of biological steps leading to cancer. It’s also key to many epigenetic…
Read MoreGenerating High-Quality Genome Assemblies from Metagenomic Sequencing
The decreasing costs in genomic sequencing over the past decade have inspired researchers to apply shotgun next-generation sequencing to entire microbial communities. While the reads generated typically cannot be assembled cleanly into individual genomes, there is often enough information produced to identify most microbes present in the population. However, this approach lacks sufficient resolution to…
Read MoreA Beginner’s Guide to Single-Cell RNA Sequencing
Since our early steps in Science we have been told that every cell in our body has the exact same genetic information (minus one or two alterations). Therefore, the great variety of cells in our body comes from gene expression – each cell must express one set of genes and repress another set to function…
Read MoreGet to Know Your Reference Genome (GRCh37 vs GRCh38)
Whether your experiment relies upon a reference-based genome assembly or mapping reads to a reference genome to identify variants, you need to choose a human reference genome assembly. But wait! You go to the FTP site of NCBI’s refseq and click on the Homo sapiens folder. There you are presented with two choices. Which one…
Read MoreCRISPR-Inspired Method Targets Large, Repetitive DNA Elements
Target capture through PCR has been a mainstay in genomics for years, but scientists working on especially repetitive, poorly characterized, or rapidly evolving regions continue to struggle to fish out those stretches of DNA for further study. However, whole genome sequencing, the only other alternative for these regions, can force researchers to pay for much…
Read MoreDe Novo DNA Sequencing and the Special k-mer
The technology for DNA sequencing was developed back in 1977 thanks to Frederick Sanger. It took a bit longer before it was possible to sequence a complete genome. This is because we needed an appropriate mathematical model and massive computational power to assemble millions or billions of small reads to a larger complete genome. Today’s…
Read MoreA Crash Course in BLAST Searching
Simple BLAST searching is pretty straightforward to many of us. Just plug in your sequence, select the species genome, and hit search! But have you ever wondered what it takes to run a BLAST query using these mammoth-sized (no pun intended!) sequence databases? BLAST searching can produce dozens, hundreds, or even thousands of candidate alignments.…
Read MoreHow to Use Ensembl to Classify Gene Variants
Even though our knowledge about genomes grows daily, and in huge leaps, we sometimes need to remind ourselves that DNA was first isolated in 1869 and its molecular structure was only identified in 1953. The PCR reaction only hit the scientific community as recently as 1983! So even though we are growing fast, we are…
Read MoreWhy You Should Use Cas9 Ribonucleoprotein Transformation for CRISPR Genome Editing
Imagine directly creating a mutation at (almost) any site in your target genome instead of screening thousands or millions of random mutants! The CRISPR/Cas9 system does just that. In its traditional form, this forward genetics approach takes 7 steps from start to mutated genome. However, there is a way to obtain your designer genome in…
Read MoreThe Good, the Bad and the Expensive of Whole Genome Sequencing
Whole Genome Sequencing (WGS) is still very cutting edge, sequencing technology and while there are a lot of perks to using it, there are also a few drawbacks. The good, the bad and the pricey are outlined below to help you navigate when it’s worth using WGS! Whole Genome Sequencing: The Good Lots of Data…
Read MoreNext Gen Sequencing Challenges
Next generation sequencing opened the doors to our genome. It gives massive amounts of information in a week – whereas Sanger sequencing takes thrice as long, and causes lab lesions due to the abusive use of pipettes. Indeed, with minimal hands-on procedures we obtain a lot of data. But nothing in Science is ever easy.…
Read MoreDNA Extraction from FFPE Tissues for NextGen Sequencing
Rapid genomic analysis offered by next generation sequencing (NGS) is ideal for personalized medicine approaches to clinical genetics, microbiological profiling, and diagnostic oncology. Many standard clinical samples are preserved as formalin-fixed, paraffin-embedded (FFPE) tissues, which presents obstacles for use in NGS analysis. FFPE tissue preservation has the benefit of keeping samples intact for histological examination…
Read MoreBest Practices for DNA Shearing for NGS
Construction of high-quality sequencing libraries is pivotal to successful NGS, and DNA quality is one of the most critical aspects of library preparation. As this Nature Methods paper illustrates, DNA shearing involves appropriate and consistent fragment sizes for sensitive and accurate sequencing, and the fragments must be accurately analyzed prior to sequencing to measure molarity…
Read MoreCodon Optimization 101
The intriguing thing about protein expression is that the combination of transfer RNAs (tRNAs) that translate the 3 letter codon into an amino acid (aa) far exceeds the number of existing amino acids (aa). If you do the math correctly, the maximum number of unique combinations using the triplet code to code for the 4…
Read MoreAn Introduction to Nanopore Sequencing
DNA sequencing is the most powerful method to reveal genetic variations at the molecular level, leading to a better understanding of our body in physiological settings, and pathological conditions. It is the beginning of the long road towards better diagnostics and personalized medicine. Even though there have been great advances in DNA sequencing technologies there…
Read MoreA Crash Course in CRISPR-Cas9 Editing in Drosophila
Get tips and tricks for performing CRISPR-Cas9 editing in Drosophila.
Read MoreHow to Design a CRISPR Cas9 Experiment and Start Genome Editing
Designing a CRISPR experiment can be daunting. We’ve got tips and pointers to help you get off on the right foot.
Read MoreNGS-Based HLA Typing Delivers More Comprehensive Information
Used for matching organ transplants to donors and other applications, human leukocyte antigen (HLA) typing is rapidly shifting from older methods to NGS technologies. This is a major step forward, as more complete views of the highly polymorphic HLA genes provide a deeper understanding of how a person’s natural genetic variation might affect transplant matches…
Read MoreHow to Test the Efficiency of your sgRNA
To successfully edit your genome of interest, one critical step is to test the sgRNA you have designed. Fortunately there are programs that have been developed such as CRISPRscan for zebrafish, SSC, Sequence Scan for CRISPR, or WU-CRISPR that you can use to predict the efficiency and the suitability of the sgRNA. However, the prediction…
Read MoreMeeting the BioPython
The Biopython Project is an amazing initiative that helps scientists use Python for bioinformatics – and it’s exceptionally easy to learn! You can access online services, parse (read) different file types, analyze, and do a bunch of fun stuff with your data with Biopython. The people behind the project have put in a lot of…
Read MoreProbability Theory and Molecular Barcodes
In biology, a molecular barcode is a characteristic DNA sequence used to distinguish and gather together similar items. Such a simple but powerful concept is useful in various applications. As an example, the Barcoding of Life project aims to identify specimens through the sequencing of standard gene regions, and use these as barcodes. On the other…
Read MoreDemystifying NGS: Depth Coverage and Deep Sequencing
NGS is not a three-headed monster. However, it can be a difficult concept to grasp—especially when you are getting started. There is a lot of new terminology, and a whole new world to discover: both in the lab bench and in interpreting your results. It helps to start somewhere. So, let’s start! Depth of Coverage…
Read MoreA Primer on Checking the Methylation State of the Genome
We all know that genes encode proteins that make up a living cell. However, the level and coordination of gene expression is really the key to the success of a living cell. One way eukaryotic cells (that’s us!) control protein expression is through addition of a methyl or hydroxymethyl group on the cytosine nucleotide. This…
Read MoreShow Disparity in Gene Expression with a Heat Map
Have RNA-seq or microarray data? What possible tools can help you find your genes of interest? Is there any pattern in your expression data? I know you are totally at sea but heat maps are now commonly used to help. A heat map is a well-received approach to illustrate gene expression data. It is an…
Read MorePhylogenetic Tree Construction Made Easy with Blast & Mega
Your DNA sequence can be put to good use fairly easily with Blast and Mega software. These programs can help in phylogenetic tree construction. You can ask questions like what is the evolutionary relationship between a set of sequences from different species? Or how have certain microbial strains arisen? Blast As any bioscientist probably knows,…
Read MoreHow to Generate High-Quality SNPs Data Sets from NGS
SNPs or single nucleotide polymorphisms are on many scientist’s wish list in experimental studies of genomic DNA sequences. Methods to detect SNPs have evolved. Now with the availability of high throughput sequencing methods, also known as next generation sequencing (NGS), SNPs can be identified in the large amounts of DNA sequence that is generated. There…
Read MoreInvestigating an Expression Quantitative Trait Locus (eQTL)
Thousands upon thousands of genetic variants are now associated with every disease and trait you can possibly think of. Such traits range from cancers to blood pressure, intelligence, height, weight… and many more! This is largely because of the advent of genome-wide association studies (GWAS). However, the vast majority of genetic loci associated with these traits are…
Read MoreVariations on the ChIP-seq Theme and Challenges of Befriending Large Datasets
ChIP-seq has proved amazing. Through these new techniques, we can obtain big datasets in a matter of days, making our lives in the lab easier and more efficient. ChiP-seq combines chromatin immunoprecipitation (ChIP) assays with whole genome sequencing. This makes it possible to understand where proteins bind to DNA and epigenetic modifications. Humans are not only their…
Read MoreGenomics Software – Doorways to Visualize Sequence Data
Are you tired of staring at all of your sequence data? Want to know the easiest way to look at it? For complex genomics data, an appropriate visualization tool is a must have. The right genomics software will make it easy-peasy to get some results as well as test all those ideas you have. Since…
Read MoreIntroduction to Linux for High-Throughput Sequencing Analysis
So, you’ve spent time planning your high-throughput sequencing experiment. You’ve chosen how many replicates to use, deliberated about sequencing depth, and kept everything RNase-free. Now you have many gigabytes of data available. What’s next? While the first step of RNA-Seq analysis is aligning your sequencing reads to a reference genome, first you need to get…
Read MoreHow to Follow up on a GWAS (Genome-Wide Association Study)
So, the genome-wide association study (GWAS) data for your disease of interest was published, and it has thrown up some very interesting associations. However, at this stage, bear in mind that this is only an association. Your project is to provide the link between the GWAS single nucleotide polymorphisms (SNP) and pathological changes. Where do…
Read MoreHow Does Automated Electrophoresis Perform DNA Size Selection?
Anytime lab processes get automated by a sophisticated scientific instrument, there can be a “black box” effect, leading users to wonder what’s going on in there. For DNA electrophoresis, it’s no different. It’s easy to see what’s happening in a manual gel, but the automated gel-based DNA size selection platforms can be more mysterious. Automated…
Read MoreAn Introduction to Shotgun Sequencing: Fire in the Hole
In the midst of all the cool new sequencing techniques and technologies out there today, you may have overlooked the tried and true method of Shotgun Sequencing. What is Shotgun Sequencing Anyway? Shotgun sequencing gets its name from the concept that a large sequence is essentially broken up in to many, many smaller pieces, similar…
Read MoreHow to Choose The Appropriate Genomic NGS Data Simulator
Let’s say that you’ve just finished gathering your NGS reads and you’re going to simulate the introduction of random mutations at specific rates into the reads. Before you move on to the next step, you need to determine which NGS data simulator will get the job done. With the ever-increasing advancement of NGS in the…
Read MoreP19 to the Rescue: How to Increase Protein Expression in Agroinfiltration
Plants are just not green gods—they can be more. You can cost-effectively express your recombinant complex proteins in a plant system. More interestingly, plants are ideal systems for producing functional monoclonal antibodies, enzymes, and vaccine components! They can also be used for protein localization studies. To save time, you can transiently express your protein using…
Read MoreDNA Sizing Tutorial: When to Use Manual Gels, Beads, and More
There are several methods for size-selecting DNA fragments prior to sequencing. How do you choose which is best? Here’s a look at various options, plus considerations to help you determine when to use each one. Manual Gels Virtually every student in a biology lab knows how to prepare and cut a manual gel—but their ubiquity…
Read MoreThe Ins & Outs of Illumina Sequencing
The future of personalized medicine depends on affordable DNA sequencing. In the race for the $1,000 genome, several sequencer manufacturers are working on making equipment that can sequence DNA and RNA faster and more accurately. But so far, only one company – San Diego, California-based Illumina – has US FDA regulatory approval to use its…
Read MoreRNA-seq: The Challenges to Diving Right In
It’s the hot new technique. With a single procedure, you can get information about all RNA transcripts at once! It sounds like a dream. While RNA sequencing (RNA-seq) has opened the door to exciting new questions, scientists interested in pursuing this technique should be aware of the roadblocks ahead of them. While RNA-seq can be…
Read MoreGenome Viewers/Editors – Three of the Best
Sometimes the wide view is the best so a good genome viewer is a must for every molecular biologist. We review three leading genome viewer packages to get you started..
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