Conducting Massively Parallel Sequencing

One of the major breakthroughs in modern biology is the development of massively parallel sequencing, also called next generation sequencing (NGS), which enabled the complete delineation of the human genome more than a decade ago. Since then many more species’ genomes have been sequenced, and the cost per genome has dropped from billions to mere thousands of dollars. New discoveries are being made as a result of the capability many research teams now possess to not only sequence chromosomal DNA, but also to identify which regions a protein of interest specifically binds (Chip-seq), analyze a whole transcriptome of a cell population under investigation (RNA-seq), or find out which RNA regions an RNA binding protein resides (CLIC-seq).

While it is inevitable that many PIs will seriously consider the inclusion of deep sequencing in their next grant proposal, it is not necessarily easy to take the first step and get their feet wet, so to speak. Knowing what format (e.g. 454 for longer reads, HighSeq for higher accuracy, or Ion Torren for bench top convenience) to use and how much to pay requires a vast amount of knowledge and experience. Even when you are done with sample prep, amplification and sequencing, to handle such massive amount of data is not trivial—transporting data alone can be a headache. A database server for storage and analysis requires another layer of expertise. There is no easy solution but to get started somehow. However, be prepared to deal with these issues.

Whether the cost on a type of next generation service is justifiable depends on whether it is required for your purposes. For example, when analyzing a person’s propensity of developing a disease by using known, disease-relevant genetic information, often times exome sequencing is sufficient. This costs anywhere between $1,000 to $3,000 with 100X coverage, significantly less than sequencing a complete genome which typically costs ~$5,000 at ~20x coverage.

High coverage sequencing of maternal blood DNA has been developed into clinically approved prenatal diagnosis of trisomy in Down’s syndrome and other chromosomal abnormalities. Transcriptome analysis helped the understanding of how reprogramming works when iPSCs are. Looking forward, with more routine use of deep sequencing we can predict with much more certainty the “off-target” effects of RNAi or cellular toxicity of chromosomal modifications enabled by ZFN, TALEN, or CRISPR. As a matter of fact, we believe that transcriptome sequencing should be required after each RNAi event to prove a specific linkage between knockdown and functions; similarly, whole genome sequencing results need to be provided after making a site directed chromosomal change in the future for high level publications.

*This blog partially resulted from discussions between Jiwu Wang and his colleagues, who are NGS experts at UCSD’s Cellular and Molecular Medicine, Moore Cancer Center, and BGI Americas.

Tags: CRISPR, deep sequencing, exome sequencing, footprint free, genomic modification, iPSCs, Massively Parallel Sequencing, NSG, TALEN, transcriptome, whole genome sequencing, ZFN

Solving the world’s problems with new biotechnology

The ability to isolate, create, synthesize, or artificially evolve living organisms towards desirable phenotypes may be increasingly important for solving many of the problems the world is facing. Such problems may include creating renewable energy using biowaste, finding biocontrol products that kill food-spoiling fungi “organically”, or assaying pathogens in the field using synthetic biological detection systems. With the arrival of synthetic biology, “it is possible to design and assemble chromosomes, genes and gene pathways, and even whole genomes”, according to the J. Craig Venter Institute. That is, if you know which genes or gene pathways you would need to put into the synthetic genome that would lead to the desired traits. So far, most published synthetic biology work involves bringing in transcription factors from a non-host source to set up an artificial network like circadian oscillators, showing that it can be done and it is interesting.

Through the process of evolution biological systems aptly self-engineer favorable traits in order to survive, but these changes require millions of years to manifest. However, there are quicker adaptations to environmental cues, such as developing antibiotic resistance, which can be achieved through a small number of mutations in hundreds or even dozens of generations. The question is how to harness this kind of adaptation for new strains that can be used as products with defined purposes? As a first requirement, you must have an assay for identifying the wanted mutants or method for augmenting their subpopulation, which is not necessarily easy and normally takes some clever designs to establish. Since evolutionary success in nature results from continuous “rounds” of gene mutagenesis, expression and selection, an evolution in the lab should ideally proceed with continuity. Previously, each round of mutation and selection takes a few days to complete. Recently, Esvelt et al. in David Liu’s lab at Harvard demonstrated one way of doing in vitro continuous evolution, by creating a lagoon of mixed E. coli and phages. By continuous dilution of the phage population through outflow, those phages that remain in the pool with properties that help them propagate in the host bacteria will have a better chance to regenerate and accumulate mutations towards the design of the assay [1].

Another aspect of natural evolution is that it occurs in a heterogeneous environment separated into niches of subpopulations with uneven stress levels. Although most evolutions with human intervention were conducted in a homologous population under the same stress and selection, a spatially complex environment may speed up evolution. This may not be easy to imagine, but if a mutant acquires some level of resistance to its environmental stress level and has a chance to move to join a population under higher stress, its relative fitness will likely increase. In addition, in a smaller population in the niche under higher stress, the mutant with marginally beneficial properties acquired under lower pressure can take over more quickly. This was demonstrated by Zhang et al. who showed that with a gradient of antibiotics applied to an array of microwells interconnected through tiny channels, new resistant strains can evolve in less than a day. Without the gradient, or separate the interconnected niches into discrete wells, no resistant populations could be obtained [2].

With more understandings like these and equipped with large scale gene synthesis, chromosome assembly, and deep sequencing technologies, we should see increasing numbers of human-made organisms serving special needs for food, health, energy, and the environment. Synthetic biology or artificial evolution won’t solve all the world’s problems, but if applied effectively and diligently, they can certainly help with many critical aspects as the technology “coevolves” with the environment.

[1] Kevin M. Esvelt, Jacob C. Carlson, & David R. Liu. “A system for the continuous directed evolution of biomolecules” Nature 499, 2011.
Qiucen Zhang, Guillaume Lambert, David Liao, Hyunsung Kim, Kristelle Robin, Chih-kuan Tung, Nader Pourmand, Robert H. Austin. “Acceleration of Emergence of Bacterial Antibiotic Resistance in Connected Microenvironments” Science 333, 2011.

New Products of the week: Modified UTP (Pseudouridine-5´-triphosphate), and Modified CTP (Methylcytidine-5´-triphosphate) for in vitro transcription of mRNA.

Promotion of the week: Friday special this week, buy 2 GFP-Trap get 1 free. Email the code “2+1GFPTrap” after placing your order of 2 GFP-Trap beads (0.25ml or 0.5ml scales only).

Tags: biocontrol, bioenergy, biofuel, biowaste, chromosome assembly, deep sequencing, food fungus, green energy, in vitro evolution, microenvironment, next generation sequencing, synthetic biology

Purifying DNA without Membrane Binding

Purifying DNA from natural samples or biochemical reactions is one the most frequently performed experiments in virtually all molecular biology labs. Binding DNA to silica membranes in chaotropic binding buffers is the currently prevailing method, pioneered by Qiagen. Before Qiagen columns and the similar columns from a number of companies, including Allele, there was the silica resin, mostly from Promega. Before silica, it was phenol extraction or CsCl gradient.

Silica-based technology has been around for more than a decade, and it is time for a new generation of technologies that are more convenient and efficient than silica membrane to take center stage of DNA purification, especially given the fast-paced advances in polynucleotide analysis in microarrays and deep sequencing. Solid Surface Reversible Binding (SSRB) technology should be a shining star in coming years. The process is simple: DNA or RNA molecules in a simple binding buffer bind to the surface of plastics of any size and shape (PCR tube, 2.0ml eppendorf, 96-well plates, even 15 ml or 50 ml conical tubes) that is treated by a special process, washed, and eluted in any volume of water or even downstream reaction buffers. The utmost convenience is that the downstream reaction can be performed in the same tube!

This process is different from the electroreversion type of binding and releasing that requires buffers of different and extreme pH. Allele Biotech has started marketing SurfaceBind PCR purification kits, and will roll out products that are specifically tailored for genomic DNA, mRNA, size-differentiated DNA or RNA, DNA or RNA from fixed samples, from different species, etc. The convenience and cost-efficiency of these systems will provide significant contributions to the broad scientific community.

New Product of the Week 101810-102410:

SurfaceBind PCR purification kit, questions? Please email us at oligo@allelebiotech.com for a product description and introduction quotes.

Promotion of the Week 101810-102410:

Updated: GFP-nAb products (equivalent to previously distributed GFP-trap), good for genomic DNA pull down by transcription factor-GFP fusion, RNA co-IP via RNA binding protein-GFP fusion. Or try our brand new, the brightest mFP–mNeonGreen and already available mNeonGreen-nAb, or anti-mNeonGreen nano antibody (also referred to by others as “nanobodies”).

Tags: deep sequencing, DNA purification, genomic DNA, microarrey DNA sample, miniprep, miRNA, nextgen sequencing, PCR cleanup, PFA linked, RNA purification, silica membrane, surface resersible binding