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Genomics at a glance – Part 2/2

NGS Workflow

First step in NGS workflow is to obtain samples for the sequencing. Either DNAs or RNAs need to be

extracted from samples. Unfortunately, sequencing technology is not at the stage that we could load

samples directly onto a sequencer. The samples have to be sheared and become random ‘short’

fragments. Also, depending on NGS platforms, DNA or RNA short fragments have to be amplified in

order to obtain sufficient amount of short DNA/RNA fragments before sequencing. This non glamorous

traditional laboratory work is still very critical for obtaining high quality sequencing results.

There are small number of different NGS platforms available currently, but Illumina sequencers are the

industry leading platform. More or less, these different platforms are based on sequencing by synthesis

(SBS) technology except Oxford Nanopore. The fundamental of SBS technology is to capture signals

when DNA polymerases add labeled complementary nucleotides on target sequences.

After a sequencer generates short sequence reads, these reads need to be mapped onto a reference

genome to figure out the origins since a sequencer generates nothing, but millions of short DNA/RNA

fragments. Adding meaningful labels on these short sequences, so called aligning, is the beginning of

NGS data analysis. This enables downstream analyses. There are many different flavor of aligners

available, but not all aligners are RNA-Seq analysis ready.

As illustrated on Figure 2, there are many ways to apply NGS in different studies. However, in terms of

target sequences, those applications can be organized into two groups, DNA-Seq and RNA-Seq. Only

about 9.2 percent of human DNA does something, and little over 1 percent of human genomes codes

Figure 1 DNA-Seq vs RNA-Seq (The original image was obtained form

https://upload.wikimedia.org/wikipedia/commons/9/91/Chromosome.gif and modified.)

Summary of content (3 pages)

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Genomics at a glance – Part 2/2 NGS Workflow First step in NGS workflow is to obtain samples for the sequencing. Either DNAs or RNAs need to be extracted from samples. Unfortunately, sequencing technology is not at the stage that we could load samples directly onto a sequencer. The samples have to be sheared and become random ‘short’ fragments. Also, depending on NGS platforms, DNA or RNA short fragments have to be amplified in order to obtain sufficient amount of short DNA/RNA fragments before sequencing.
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proteins. And, these coding regions are shared by about less than 20,000 genes. Hence, a small fraction of chromosome sequences transcribed to mRNAs in the nucleus of cell as shown in Figure 5. Once preRNAs are synthesized, splicing mechanism process removes introns and rejoins exons to create messenger RNAs (mRNAs). These matured RNAs are called messenger RNAs since they are transported out to cytoplasm to deliver genetic information to protein synthesis apparatus located in cytoplasm.
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between two cells under different conditions. For example, RNA-Seq is useful to compare gene expressions between normal cells and cancer cells from a patient. Limitations of NGS Neither DNA-Seq nor RNA-Seq alone can provide a complete answer for any biological question. This is not only NGS’s problem, but also any experimental process or single technology in life sciences. A conclusion drawn from a controlled experiment will not actually reflect the real conditions in a life form.