The Human Genome Project was declared complete in 2003. The entire genome of an organism consists of only four deoxyribonucleotide bases – A, T, G and C. Determining the sequence of the four rather simple molecules within approximately 3 billion base pairs was quite a challenging task for the team of brilliant researchers determined to sequence all the genes within the human genome.

DNA sequence analysis can involve multiple steps that include the alignment of DNA sequences, and searches against established databases. Sequence analysis can provide the research team with an understanding of the function of different genes, their interactions with protein, structures, and evolution. Comparison of the new sequences with the already stored DNA sequence data allows researchers to explore the similarity between the genetic makeup of organisms and their differences.

In molecular biology, DNA sequence analysis is instrumental in the:

i. Comparison of different DNA sequences to find the similarities and relationship between them (identification of homologs).
ii. Documentation of differences between comparable sequences to determine single nucleotide polymorphism (SNP) and genetic markers.
iii. Determination of active sites, sites of post-translational modifications, reading frames, intron and exon distributions, regulatory elements of a gene, and other intrinsic features.
iv. Studying the evolution and genetic diversity in organisms (prokaryotes and eukaryotes).
v. Identification of the structure of macromolecules from the complete sequence analysis.
vi. Creation of phylogenetic trees using DNA sequences from multiple organisms.
vii. Determination of DNA sequences from unknown or previously uncharacterized microorganisms within their natural habitat.

The first step in the analysis of DNA: first-generation DNA sequencing

The first generation of DNA sequencing allowed the team to sequence the entire genome. While using Sanger sequencing, researchers typically sequenced DNA molecules of up to 900 base pairs routinely. The analysis of the resulting DNA containing the labeled ddNTP from the Sanger sequencing involves the use of a gel matrix called the capillary gel electrophoresis. The original DNA sequence can be reconstructed from the different dye-labeled molecules registered by the sensor, one after the other. The final data shows a series of peaks of fluorescence intensity.

Next-generation sequencing: faster and cheaper DNA seq analysis

Large genome sequencing projects that require a quick turnaround demand a method that is faster than Sanger sequencing.

When the question of sequencing entire genomes and metagenomes of organisms arise, the only viable answer is Next Generation Sequencing (NGS). NGS has several applications in biotechnology, molecular biology, biomedicines, microbiology, oncology, and genetics. While there is a wide range of NGS techniques now available, almost all of them have these few traits in common.

i. Microscale – multiple reactions can be conducted on a single chip.
ii. Highly parallel – one of the most significant advantages of NGS is that you can conduct multiple sequencing reactions simultaneously.
iii. Lightning-fast – since you can conduct multiple reactions simultaneously, you will save significant time.
iv. Cost-efficient – it is significantly cheaper than Sanger sequencing.
v. Shorter read – the read length typically ranges between 50 and 700 nucleotides.

The evolution of sequence alignment software has made the pairwise alignment and multiple sequence alignment of DNA sequences faster and easier than ever. The set of tools or software can align nucleotide sequences in pairs or among three or more sequences in parallel.

What role does sequence alignment software play in DNA seq analysis?

Pairwise alignment

Pairwise alignment is necessary to find the best alignment between the target sequence and a recognized template DNA sequence. All pairwise alignment methods that are currently in use have trouble in aligning repetitive sequences like the microsatellite regions of DNA.

Multiple sequence alignment

The variation in the currently available suite of software for MSA provides room for maximizing scores and best-fit of alignments during DNA seq analysis. The alignment of multiple short sequences is often necessary after a round of NGS. It leverages progressive alignment that uses the pairwise alignment procedures iteratively to align sequences in order of the closest matches to the least matching ones.

With the development of complete DNA seq analysis software platforms, alignment and analysis of sequence data from whole genome sequencing and whole exome sequencing studies have become easier, faster and more accurate than they were in the early 2000s.

How does automated software make DNA seq analysis quick and easy?

When there was no one consolidated platform for sequence alignment, analysis, QC, and visualization, second generation DNA sequencing used to be a cumbersome process even for the best-equipped laboratories. Right now, the presence of wholesome automated DNA seq analysis platforms allow the complete alignment and analysis of DNA sequencing data within a couple of hours or less has made NGS a ubiquitous part of most genome studies.

Whether a team requires a pairwise alignment or a multiple sequence alignment, the automated software platforms with user-friendly APIs can deliver. The state-of-the art DNA seq analysis platforms promise ready-to-print reports, along with audit-friendly formats from simple NGS seq data formats. You can also check the coverage of your data with respect to the whole genome/exome, browse its variant summary and visualize the genome coverage for your NGS data sample. It is a fast and cost-efficient process trusted by hundreds of eminent research teams working on biomedical sciences, genetics, and metagenomics.

The Human Genome Project was an international project founded in 1990 that continued for 13-years, and it cost around $3 billion. In 2001, the cost of sequencing the human genome was reduced to $100 million. Today, sequencing the entire human genome using NGS technique would not cost more than $1400, and it would only take one day or two, depending on the particular technique the research group uses.

The easy availability of DNA seq analysis software also plays a critical role in the popularity of NGS. The advancement of sequencing and alignment techniques now allows the research teams to receive a detailed result online via interactive and print-ready reports. The easy availability of commercial alignment and analyses software enables researchers to edit the reports using sliders (for adjusting parameters) and filters and share the reports online with their peers.