Genomics and Epigenetics
A Colorful Route to Sequencing Success
An oft-repeated maxim in biological bench science is that any experiment is only as good as its control. A control is an unchanging standard of comparison in an experiment, and an internal control is typically a standard reaction run together with the test reaction in the same reaction mixture. The purpose of an internal control…
Read More CRISPR Multiplexing Strategies: What’s The Choice?
Although multiplex CRISPR gene editing can be accomplished by simply introducing more than one gRNA to your target cells, there are many alternative — and more efficient — ways of achieving this goal. This article discusses these alternative CRISPR multiplexing strategies and highlights their potential caveats. Not sure whether multiplex CRISPR gene editing is right…
Read More Multiplex CRISPR Gene Editing: What’s It For?
The RNA-guided nature of CRISPR nucleases (e.g., Cas9, Cpf1) makes them highly amenable to multiplex applications, including multi-gene knockout and large-scale genomic modifications. This article — part 1 of a two-part series on multiplex CRISPR gene editing — discusses these applications and outlines practical considerations for multiplex CRISPR gene editing experiments. New to gene editing…
Read More Dead Useful: CRISPR-Cas9 Epigenome Editing
Given the rapid pace with which genome editing technologies have been developed and adopted, it comes as no surprise that the original CRISPR-Cas9 system has been successfully modified by some very clever scientists. No longer are we limited to the ‘simple’ case of editing the genetic sequence of your biological system of choice, but there…
Read More DNA Extraction for Next Gen Sequencing
The advent of Next Gen Sequencing (NGS) has been truly amazing. One of the marvels that is often overlooked is how advances in DNA extraction technology have helped streamline NGS workflows. The original standard – phenol/chloroform extraction – is not well suited to the automated nature of today’s sequencing workflows (though with the emergence of…
Read More Get Prepped: Nanopore Library Preparation Optimization
Nanopore is a relatively new sequencing platform and researchers are still trying to optimize the protocol for their own specific applications. In our lab, we work primarily with metagenomic samples and use the 1D sequencing kits. Over the past year, we have optimized this technique. To check the quality of the Nanopore library preparation we…
Read More CRISPR-Based Activation (CRISPRa) of Genes: A How-To Guide
The purpose of this article is to walk you through the steps needed to overexpress genes using CRISPR/Cas9-based activation (CRISPRa). A broader overview of this topic (including CRISPR-based repression) can be found here. CRISPR/Cas9 is more than a programmable nuclease. When stripped of its nuclease activity, it can activate and repress transcription, alter chromatin structure,…
Read More CRISPR-Cas9 Genome Editing: Weighing the Pros and Cons
Genome editing is a hugely powerful tool which can help you to address a multitude of questions in your research. However, it is not necessarily the best tool for the job in every situation. Below is a discussion of the main advantages and disadvantages associated using CRISPR-Cas9 for genome editing. The Pros It’s Simple to…
Read More What’s that Organism? Using DNA Barcoding for Species Identification
In both the lab and field, it is important to know what species we are working with. While morphological data has always been a tried and true method of identifying species, DNA barcoding allows us to identify species when we don’t have that option (e.g. if we don’t have enough of a specimen to identify…
Read More How to Confirm Your CRISPR-cas9 Genome Editing Was Successful
You have sweated at the lab bench and in the cell culture suite for weeks (or more likely months) to plan and optimize for successful genome editing. Finally, you’ve got the guide RNAs and the vectors into the cells. Yes! But your work is not yet finished. You can’t take for granted the fact that…
Read More Use ddRAD-seq to Study Non-Model Organisms
Reduced-representation genome sequencing has been one of the most important advances in the last several years for enabling massively parallel genotyping of organisms for which there is no reference-grade genome assembly. An implementation of the approach known as ddRAD-seq, first conceived in the Hoekstra lab at Harvard, has been widely adopted by the plant and…
Read More Reducing GC Bias in WGS: Moving Beyond PCR
WGS technologies have seen significant progress since the completion of the Human Genome Project in 2003. First-generation Sanger Sequencers were limited by lengthy run times, high expenses, and throughputs that read only tens of kilobases per run. The arrival of second-generation sequencers in the mid-2000s brought about the plummeting of sequencing costs and run times,…
Read MoreHow to Improve Your WGS DNA Library
In whole genome sequencing (WGS) initiatives it is not enough to simply sequence the whole length of the genomic DNA sample just once. This is because genomes are usually very large. The human genome, for example, contains approximately 3 billion base pairs. Although sequencing accuracy for individual bases is very high, when you consider large…
Read More Generating RNA-seq Libraries from RNA
One of the most powerful methods of modern cellular biology is creating and analyzing RNA libraries via RNA-sequencing (RNA-seq). This technique, also called whole transcriptome shotgun sequencing, gives you a snapshot of the transcriptome in question, and can be used to examine alternatively spliced transcripts, post-transcriptional modifications, and changes in gene expression, amongst other applications.…
Read More Battle of the Methods: Whole Transcriptome Versus mRNA-seq
Maybe you want to examine the entire transcriptome or maybe you want to investigate changes in expression from your favorite gene. You could do whole transcriptome sequencing or mRNA-seq. But which one is right for your project? From budget considerations to sample collection, let’s briefly look at both to see which might be best for your…
Read More Analyzing RNA-Seq Data
RNA-seq is based on next-generation sequencing (NGS) and allows for discovery, quantitation and profiling of RNA. The technique is quickly taking over a slightly older method of RNA microarrays to get a more complete picture of gene expression in a cell. Data generated by RNA-seq can illustrate variations in gene expression, identify single nucleotide polymorphisms…
Read More Kick-Start Your Gut Microbiome Study in Four Easy Steps
Today, the gut microbiome is garnering a large amount of media attention for its role in human health and disease. From influencing immune responses to impact our brain, the gut microbiome is an important and necessary aspect of our life. So much so, that current investigations in the gut microbiome are focusing on developing biomarkers for…
Read More Studying the Epigenome by Next Generation Sequencing
The epigenome has been in the research spotlight, and for good reason. Not only has it been associated with the developmental stages of an organism, but epigenetic alterations lead to disorders and have been linked to many human diseases. So, the question stands: what exactly is an epigenome? What Is the Epigenome? Simply put, the…
Read More 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 More ChIP-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…
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