DNA sequencing

High-throughput sequencing allows us to achieve our objective, which is to characterize the resistance profiles of microbial communities. Nowadays, this can be performed reliably and at a fraction of the price from few years ago. The history of DNA sequencing is truly fascinating and one of the milestones of science. We highly encourage you to read more about it to really understand all the challenges that scientists had to overcome so we could be where we are today. Here you can find nice description of these historical events.

However, now that samples are extracted, they should be prepared before being sent to sequencing. This step is often done in the sequencing centers themselves, but many researchers also choose to prepare their own samples for reasons such as to maintain the consistency with the handling performed during the extraction process performed previously (ideally the same operator). This can also significantly reduce the direct costs involved with the project as well.

The type of sequencing that will be performed and which technology will be employed are important factors here. Thus, it is highly encouraged that scientists get in touch with their sequencing center of choice as early as possible. Important factors to consider here are, for instance, the purpose of the sequencing and the length of the desired reads. Even though the use of long-read technology such as PacBio or Nanopore has been steadily increasing, for metagenomic sequencing short-reads, such as the ones processed in Illumina machines, are often utilized.

With made decisions as to which technology will be used, library preparation will allow the referred DNA sample to become ‘readable’. This process is often divided into three different steps: fragmentation, end-repair, and ligation to adapters. Let us go through each of them now:

Fragmentation – If short-read technologies are employed, such as Illumina, then the DNA needs to be fragmented into small pieces so it can be processed in the sequencing machine. The length of these fragments to be processed in this technology could be, for instance, ~300 base pairs. For other long-read options, such as PacBio or Nanopore, the length of each fragment can be much longer up to 15-20 kilo bases. The step of fragmentation can be performed with two different approaches: physically or enzymatically. Although physical fragmentation requires the availability of specialized equipment, such as an ultrasonic bath (or probe), it is generally observed it induces less sequence-specific bias compared to enzymatic shearing of DNA. The efficacy of the fragmentation can be checked by running a small amount of the sample in gel electrophoresis.

End-repair – Fragmentation results in a variety of heterogeneous fragments containing overhangs. End-repair will remove the overhangs and additionally it will add an adenosine to the 3’ end of the fragment. This ‘A-tailing’ step will facilitate the ligation step, and will also reduce the chances of fragments ligating to each other as opposed to the adapters.

Ligation – Now that the fragments are ready, the adapters need to be ligated to them by an enzyme. Adapters are DNA sequences specific to the technology platforms in which they will be sequenced. Their incorporation allows for binding of each fragment to the flow cells of the sequencing machine. Each adapter will have a T-base overhang, thus complementing the ‘A-tailing’ step described just above.

There are a variety of commercial kits available that incorporate all this steps and allow the researcher to tailor their choices according to the needs of the project and the characteristics of the referred sample. Many of these kits now incorporate fragmentation and ligation into one step called tagmentation.

An additional optional step to the process is PCR amplification of the adapter-ligated fragments. This approach allow the enrichment of successfully ligated adapter-fragment sequences, and increases the library size. However, caution must be exercise as additional rounds of amplification can introduce a bias to the analysis. In the cases where PCR amplification is necessary, a control sample without amplification is recommended when possible.

Now the sample is ready to be sequenced! What happens next? Follow us to the next step!

References

Heather JM, Chain B. The sequence of sequencers: The history of sequencing DNA. Genomics. 2016 Jan;107(1):1-8. doi: 10.1016/j.ygeno.2015.11.003. Epub 2015 Nov 10. PMID: 26554401; PMCID: PMC4727787.