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DNA sequencing made simple

Get high quality results, quickly and easily, for whatever type of DNA you need to sequence.

DNA sequencing made simple

Widely used worldwide, Sanger sequencing is the Gold Standard in DNA sequencing due to its high accuracy on very long read-lengths using a small amount of target molecules. Next-generation sequencing (NGS) came, well, next which brought the power of sequencing an entire genome in a couple of days. The sequence can be less accurate due to errors introduced by lack of depth and coverage, which are often compromised due to the high price of NGS. The reads are shorter than in Sanger sequencing, so can be challenging to align to the known reference genome with confidence. What we typically see now is that NGS casts a wide net across the genome and Sanger sequencing is used to make sure we have the right fish, i.e. to validate each novel sequence.

Sanger sequencing

It was used to sequence the Human Genome but now it is used for validation of novel sequence from Next-Generation Sequencing (NGS) platforms, confirming cloned sequences, and for quality control in the pharmaceutical industry. The process of growing colonies, purifying the DNA prior to the Cycle Sequencing reaction, then purifying again before the Sanger sequencing run is still regarded as time-consuming and tedious.

One challenge of Sanger sequencing involves the robust acquisition of high quality reads and long read-lengths using a simple DNA purification workflow. Using our workflow, pass rates above 96% with read-lengths of >650bp are regularly achieved using nearly half the steps of the traditional workflow.

Starting with a ligation reaction, which doesn’t even need to be especially high-yielding, the addition of TempliPhi for isothermal rolling circle amplification (RCA), will generate micrograms of high quality DNA. TempliPhi amplifies circular DNA (plasmids) very efficiently, using random primers to bind and guide the polymerase reaction to replicate around and around the circle, spinning out long stretches of DNA.

The product of TempliPhi can be used directly in the Cycle Sequencing reaction. Once that reaction is finished, a simple purification step to remove unincorporated dyes and nucleotides by gel filtration yields Sanger sequencing-ready template. Specially formulated for Sanger sequencing, AutoSeq gel filtration columns or the high-throughput version, AutoScreen, in 96-well plate format, are pre-equilibrated in double distilled water, so that the finished product to be loaded onto the Capillary Electrophoresis (CE) instrument will not have any salts which can compromise Sanger sequencing results.

Workflows for different types of sample and sequencing

Figure 1. Workflows for different types of sample and sequencing

Difficult template

GC-rich templates can form structures which degrade Sanger sequencing results. For this common problem, TempliPhi Sequence Resolver was developed. Used just like regular TempliPhi, this specially formulated kit resolves the issues posed by GC-rich templates.

Next-generation sequencing

The easiest part of NGS is hitting the “Sequence” button on your HiSeq. The challenging parts are all the steps that lead up to hitting that button. There are several platforms with their own unique challenges, but we will focus on the most widely used one – illumina platforms.

Complete sequence can only be obtained from small fragments of double-stranded DNA (typically 200-500 bp in size) because of the special chemistry and unique flow-cell of illumina platforms. A target genome must be broken up into usable pieces of specific size and manipulated to ensure that all of the target genome is represented in the pieces that are bound to the flow-cell.

The first step in the library prep process is fragmentation of the target genome by mechanical or enzymatic shearing, followed by end-repairing/A-tailing, then ligating adaptors. For proper attachment to the flow-cell, each of two adaptors must be ligated to every fragment of DNA – one at each end – followed by several rounds of PCR using the adaptor sequences as forward and reverse priming sites. Magnetic bead clean-up and subsequent elution yields sequence-ready fragments that are bound to the flow-cell.

As you may guess, with each step of the library prep process there is the possibility for template loss, potentially resulting in gaps in the final sequencing coverage. Those missing sections could include an important single or multi-nucleotide variant, insertion/deletion (“Indel”) or transposed element that may hold the key to a disease pathway. To limit gaps and obtain complete genomic coverage, researchers sometimes increase the depth – the amount of times each section is sequenced – e.g. 30X, 100X, etc., but the results are limited by the quality of the starting material attached to the flow-cell. And on top of that, in many cases, template DNA is limiting and difficult to obtain. To obtain the most complete coverage across the whole genome, or even just the whole exome, it is essential that the template gDNA is in abundance, with as much sequence redundancy across the genome to compensate for the losses during library prep. So, how can this be achieved?

Phi29 whole genome amplification

The discovery that the reaction of the high fidelity Phi29 DNA polymerase of RCA works on linear DNA as well as circular DNA opened-up the world of whole genome amplification. This is called multiple-strand displacement amplification (MDA). Suddenly, the very small amount of full-length human genomic DNA in a cell could be amplified, yielding micrograms of DNA from nanograms. This process enabled numerous downstream analyses from a single sample: PCR, qPCR, microarray, and of course, sequencing. Making bulk amounts of DNA allows researchers to start with more DNA and decreases the chance of having gaps in NGS coverage. The use of the MDA reaction with our GenomiPhi kits prior to NGS library prep is standard in any sequencing lab. GenomiPhi can, of course, be used prior to Sanger sequencing, too. Because the amplification of linear DNA using GenomiPhi is slightly less efficient than RCA, requiring multiple binding sites across the genome to support amplification, it works best if the template is greater than 1 kb.

Better extraction methods

Spin column extraction kits became popular in the 1990s because they were easy and safer to use than the standard Phenol:Chloroform method. The principle of these kits involves DNA or RNA binding to a column, the unwanted material is washed away, then finally the DNA or RNA is eluted. However, the multiple spins, applications of buffers, as well as the efficiency of both the initial binding of the desired template and the final elution, can limit the final amount of template obtained as well as the genomic coverage. In addition, all the manipulation can fragment and damage the template. So, even though Phenol is dangerous to use and even prohibited at many research sites, researchers are returning to Phenol:Chloroform, because the quality and length of template obtained is unmatched.

Other challenges are posed by the source of the DNA used. For plants and seeds, the challenge is removing all the polysaccharides without damaging the template. For cancer researchers, extracting DNA from Formalin-fixed Paraffin-embedded (FFPE) tumor samples, which is essential to trace a possible tumorigenic mutation to archived tumors, is difficult.

What researchers need is a kit that can give them the quality and length of template of Phenol:Chloroform without Phenol, geared toward their target material; enter the Nucleon product range. Nucleon kits offer the ease-of-use, quality and length of template of Phenol:Chloroform without Phenol. In addition, there are kits designed specifically for plant and fungal material called PhytoPure, for Blood & Cultured Cells called BACC, and a third for Hard Tissue and FFPE, simply named Nucleon HT.

You may have already guessed that the DNA extracted with Nucleon is perfect for GenomiPhi whole genome amplification. This gives the added assurance that the researcher needs to reduce those sequencing coverage gaps as much as possible.

Single Cells or Trace Amounts of DNA Template

Up to now our knowledge of the genome has been limited to populations of cells. The variation in genome sequence between cells was impossible to characterize. You may ask, "why would we need to know those differences?" One example is the formation of a tumor. Somatic mutations randomly occur when cells undergo mitosis. Most somatic mutations occur in a part of the genome that will have no downstream effect on gene expression or the mutation will trigger the cell to die. A tumor generally starts from the accumulation of somatic mutations. To understand tumorigenesis, is to understand when and where a tumor will form. To characterize tumorigenesis, the heterogeneity of a tumor must be characterized. With our ability to isolate single cells, lyse them to release their genome and sequence it by NGS, we come closer to the possibility of predicting where and when a tumor will form. The challenge here is the amount of DNA: a single human cell only has about 7 picograms of DNA. That’s where Single Cell GenomiPhi is essential. This kit lyses the cell and amplifies its entire genome in a single tube in about two hours. The typical yield is 4 to 7 micrograms, which is more than enough for whole genome sequencing.

gDNA amplified with Single Cell GenomiPhi DNA Amplification Kit results in a high percentage of exome sequence coverage when run in whole exome sequencing (10x coverage)

Figure 2. gDNA amplified from 5 human cells using Single Cell GenomiPhi DNA Amplification Kit results in a high percentage of exome sequence coverage when run in whole exome sequencing (10X depth).

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