Next Generation Sequencing - A Step-By-Step Guide to DNA Sequencing.

ClevaLab. The Human Genome Project uncovered  all 3. 2 billion bases of the human genome.

This project started in 1990 and took until 2003  to complete 85 percent of the first genome. But, in 2022, the gaps got filled and the sequence  became complete. So in total, sequencing the human genome took 32 years.

Now, with Next  Generation sequencing or NGS, it takes only a day to sequence a person's entire genome. One  day is a dramatic speed increase compared to 32 years! The difference is due to the number of DNA  strands sequenced at once.

Billions of DNA strands get sequenced simultaneously using NGS. However,  only Sanger sequencing was available for the Human Genome Project. With Sanger Sequencing, only  one strand can get sequenced at a time.

However, NGS only works because the Human Genome Project  created a human reference DNA sequence. The basic principle behind NGS is that DNA can be cut  into small pieces and sequenced. The sequences of these small pieces then get assembled based on the  reference genome.

NGS can be used to sequence both DNA and RNA. First, samples get collected, and  the DNA or RNA gets purified. Next, the DNA or RNA gets checked to ensure it's pure and undergraded. 

RNA first needs to be reversed-transcribed into DNA before it can get sequenced. A library  then gets prepared from the DNA. A library is a collection of short DNA fragments from a long  stretch of DNA.

Libraries get made by cutting the DNA into short pieces of a specified size. This  cutting gets done by using high frequency sound waves or enzymes. Then sequences of DNA called  adapters get added to each end of a DNA fragment.

These adapters contain the information needed for  sequencing. They also include an index to identify the sample. Finally, any non-bound adapters get  removed, and the library is complete.

Depending on the application, there can be a PCR step to  increase the library amount. A successful library will be of the correct size. It will also be of  a high enough concentration for sequencing.

The main sequencing instruments used in NGS are from  Illumina. These instruments use a method called sequencing by synthesis. The sequencing occurs  on a glass surface of a flow cell.

Short pieces of DNA, called oligonucleotides, are bound to the  surface of the flow cell. These oligonucleotides match the adapter sequences of the library. First,  the library gets denatured to form single DNA strands.

Then this Library gets added to the flow  cell, which attaches to one of the two aligos. The strand that attaches to the oligo is the forward  strand. Next, the reverse strand gets made, and the forward strand gets washed away.

The library  is now bound to the flow cell. If sequencing started now the fluorescent signal would be too  low for detection. So each unique library fragment needs to get amplified to form clusters.

This  clonal amplification is by a PCR that happens at a single temperature. Annealing, extension and  melting occur by changing the flow cell solution. First, the strands bind to the second oligo on  the flow cell to form a bridge.

The strands get copied. Then these double-stranded fragments  get denatured. This copying and denaturing repeats over and over.

Localized clusters get  made, and finally, the reverse strands get cut. These strands get washed away, leaving the  forward strand ready for sequencing. The sequencing primer binds to the forward strands. 

Next, fluorescent nucleotides G, C, T and A get added to the flow cell along with DNA polymerase.  Each nucleotide has a different color fluorescent tag and a terminator. So only one nucleotide can  get sequenced at a time.

First, the complementary base binds to the sequence. Then the camera reads  and records the color of each cluster. Next, a new solution flows in and removes the terminators. 

The nucleotides and DNA polymerase flowing again, and another nucleotide gets sequenced. These read  cycles continue for the number of reads set on the sequencer. Once complete, these read sequences get  washed away.

Then the first index gets sequenced, and washed away. If only a single read is needed,  the sequencing ends here. But, for paired-end sequencing, the second index is sequenced, as  well as the reverse strand of the library.

There is no primer for the second index read. Instead, a  bridge gets created so that the second oligo acts as the primer. The second index is then sequenced. 

These two index reads use unique dual indices. These allow the use of up to 384 samples in the  same flow cell. Next, the reverse strand gets made, and the forward strands are cut and washed  away.

The reverse strands are then sequenced. Once the sequencing is complete, any bad reads  get filtered out. These include the clusters that overlap, lead or lag with sequencing or are of low  intensity.

The clusters cannot overlap on a patent flow cell, but there can be more than one library  fragment per nanowell. These polyclonal wells will also get filtered out. Next, the reads passing the  filter get demultiplexed.

Demultiplexing uses the attached indexes to identify and sort reads from  each sample. Finally, the reads get mapped to the reference genome. The different reads align to  the reference genome, overlapping each other.

Paired-end sequencing creates two sequencing reads  from the same library fragment. During sequence alignment, the alogarithm knows that these reads  belong together. Longer stretches of DNA or RNA can get analyzed with greater confidence that the  alignment is correct.

Read depth is an essential metric in sequencing. Read depth is the number  of reads for a nucleotide. Average read depth is the average depth across the region sequenced.

For  whole genome sequencing, a 30x average read depth is good. A 1500x average read depth is suitable  for detecting rare mutation events in cancer. Another essential metric is coverage.

The aim is  to have no missing areas across the target DNA. NGS gets used in a wide variety of applications.  In diagnosing cancer and rare disease, treatment guidance for cancers, and many research areas from  ecology to botany to medical science.

Both DNA and RNA can be sequenced. It could be the whole genome  or transcriptome, just the coding regions (called exomes) of the DNA, or target genes in the DNA or  RNA. All types of RNA can be sequenced including non-coding RNAs such as microRNAs and long  non-coding RNA.

In addition, cell-free DNA, single cells, as well as methylation or  protein binding sites can also get sequenced.