Next Generation Sequencing (NGS) is a useful tool in determining the DNA sequence, information
which is valuable in furthering our understanding of biological processes. Compared to other
tools, NGS is flexible and it can be used in different applications, including sequencing
the exome and methylated cytosines in the genome. However, there are parameters to be
considered prior to running an NGS experiment. For all your NGS experimental needs, Applied
Biological Materials, or abm, offers a wide range of affordable services, including whole
genome sequencing, RNA sequencing, exome sequencing, and even lane rentals.
In our previous video, we discussed how to prepare samples for sequencing. Here, we will
outline the quantitative and qualitative measures needed to ensure a good sample for sequencing.
We invite you to watch our previous video before starting this one.
Although the current NGS platforms available on the market are very accurate, they are
still prone to error. Even at accuracies of 99% and greater, a sequence generated may
contain incorrect nucleotides. This means that if a machine’s accuracy is 99%, one
base pair is read incorrectly out of 100. Since NGS platforms generate high amounts
of output, these errors can add up quickly. The way to circumvent NGS platform limitations
is to sequence nucleotides multiple times. The number of times a nucleotide is sequenced
is referred to as “coverage” or “depth.” Coverage per genome can be calculated by dividing
the total output generated in a sequencing run by the total size of the sample sequence.
For example, running a human genome, which is approximately 3 billion base pairs, will
yield approximately 333X coverage on the Illumina High Seq 2500, which has a max output of 1000
giga-base pairs. Consult abm’s NGS experts for the appropriate coverage of your experiment
here. For detailed information on the appropriate coverage required for different NGS applications,
please view our knowledge base here. Prior to sequencing, the sample library must
be validated quantitatively and qualitatively to verify if there is a sufficient amount
of good quality DNA. What is considered a good quantity of DNA depends on the library
protocol’s specifications. Having either more or less DNA results in less efficient
sequencing reaction runs. This generates low quality data due to read problems from flow
cell saturation, or reduced coverage because of insufficient DNA. In terms of quality,
a good quality library is one that has a diverse set of DNA fragments with minimal duplicate
fragments. This is important because during PCR amplification, duplicates of fragments
will be generated. The consequence of duplicate fragments is that the sequencing reaction
will be biased towards these fragments. Rather than having a wide range of fragments sequenced,
the same fragments are sequenced repetitively, resulting in overrepresentation in the machine
output. Library quantification is performed using
either qPCR or a fluorometric method like Qubit. Some libraries may only be quantified
using one of the two methods. Sample library quality is then verified with the Bioanalyzer.
qPCR is a method of quantifying a sample library before sequencing. It is ideal when there
is an insufficient amount available for fluorometric quantification, commonly due to no PCR amplification.
It is also a more sensitive way, relative to Qubit, to quantify the adapter-ligated
fragments in a sample. qPCR selectively amplifies such fragments, so it avoids the inaccuracies
of Qubit that result from being unable to distinguish between fragments which can and
cannot be sequenced. The only drawback to this procedure is that it is very time-consuming.
Qubit is an alternative to qPCR for quantifying a sample library. Relative to qPCR, it provides
results faster; however, it is not applicable for cases where there is no PCR enrichment
as it is less sensitive than qPCR and requires more sample. Quantification is performed by
mixing the sample, which may be diluted, with the appropriate dye, which is then illuminated
and detected by the machine. Note that a standard must be measured with the appropriate assay
prior to sample quantification. The Bioanalyzer is used to check the size
distribution of the library before the sequencing reaction, including whether the sizes selected
during sample library preparation are present. The Bioanalyzer is a machine that reads gel
chips containing samples in the wells. The chips are similar to agarose gels, except
in a smaller format. The protocols for DNA and RNA are similar to each other. The first
step is to introduce the gel into the chip and pressurize it; this will evenly distribute
the gel, minimizing errors in machine analysis later on. Once complete, markers, ladders,
and samples (either diluted or undiluted) are loaded onto the chip. There may be additional
reagents needed depending on the kit requirements. The chip is then vortexed before it is loaded
onto the Bioanalyzer. The machine will monitor each well for sample. This is visualized with
peaks on a graph. The location of the peaks will indicate the markers and the sample size
distribution of the library, while the peak height shows the amount of fragments at a
specific size. As part of abm’s NGS service, we perform quality control all sample libraries
to ensure valid sequencing data are delivered to our customers.
Please leave your questions and comments below and we will answer them as soon as possible.
Thank you for watching!