Next Generation Sequencing

The Human Genome Project was a scientific endeavor that aimed to uncover the 3.2 billion bases of the human genome. Initiated in 1990, the project took until 2003 to complete 85% of the first genome. It successfully sequenced the entire human genome, thereby providing a complete set of genetic instructions for constructing, maintaining, and operating a human being. With Next Generation Sequencing (NGS) technology, it now only takes a single day to sequence the entire human genome. NGS permits the sequencing of billions of DNA strands at once.

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NGS works by cutting DNA into small pieces and sequencing the sequences of the small pieces that make up a sample based on the reference genome. NGS can be used to sequence both DNA and RNA. The sequencing process begins with the collection of the sample, which then undergoes a purification process to ensure the purity and integrity of the RNA and DNA. RNA is reverse-transcribed into DNA before it can be sequenced. The DNA library is then prepared, which contains short fragments of DNA. This is accomplished by cutting DNA into short pieces of a specific size using enzymes or high-frequency sound waves. Adapters containing the information required for sequencing, as well as an index to identify the sample, are added to each DNA fragment. Once the non-bound adapters are removed, the library is completed. A successful library will have the correct size and a high enough concentration for sequencing.

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The instrument used for analysis is known as ILLUMINA SEQUENCING BY SYNTHESIS (SBS). Sequencing occurs on the glass surface of a flow cell. Short pieces of DNA called oligonucleotides are bound to the surface of the flow cell. These attached oligonucleotides match the adapter sequence of the library. Initially, the library is denatured to form a single DNA strand. Then, the library is added to the flow cell, which binds to one of the two oligonucleotides. These strands that attach to the oligonucleotide are known as forward strands. Next, the reverse strand is generated, and the forward strand is washed away. The library is now bound to the flow cell; if sequencing started now, the flow cell signal would be too low for detection. Thus, each unique library fragment must be amplified to form clusters. Clonal amplification is accomplished via PCR. Annealing, extension, and melting occur by altering the flow cell solution. The first strand binds to the second oligo in the flow cell to form a bridge. The strand is copied, and these double-stranded fragments are denatured. This copying and denaturing repeat over and over until a localized cluster can be made, and finally, the reverse strands are chopped. These strands are washed away, leaving the forward strand ready for sequencing. The sequencing primer binds to the forward strand, and fluorescent nucleotides G, C, T, and A are added to the flow cell along with DNA polymerase. Each nucleotide has a different color fluoresce tag and terminator, so only one nucleotide is sequenced at a time.

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First, the complementary base binds to the sequence, and then the camera reads and records the color of each cluster. Next, a new solution flows in and removes the terminators. The nucleotide and DNA polymerase flow in again and another nucleotide is sequenced. These read cycles continue for the number of reads set on the sequencer. Once complete, the read sequences are washed away, and then the first index is sequenced and washed away. If only a single read is required, then sequencing ends here. After 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 is created so that the oligonucleotide can access the primer. These two indices reads utilize unique dual indexes, allowing the use of up to 384 samples in the same flow cell. Next, the reverse strand is generated, and the forward strand is cut and washed away. Reverse strands are then sequenced. Once sequencing is complete, bad reads, including overlap, lead or lag sequencing, or those of low intensity, are filtered out. Clusters cannot overlap on a patterned flow cell, but there can be more than one library fragment per well. Next, the reads passing in the filter are demultiplexed. Demultiplexing uses the attached indices to identify and source reads from each sample.

Finally, the reads are aligned to the reference genome. The different read alignments to reference genomes overlap each other. Paired-end sequencing produces two sequencing reads from the same library fragment. During sequence alignment, the algorithm recognizes that these reads belong together. Longer stretches of DNA and RNA are analyzed with greater confidence that the alignment is accurate.

Read depth and coverage are critical metrics in sequencing. Read depth is the number of read nucleotides. The average depth is the average depth across the region sequenced. For whole-genome sequencing, a 30* average read depth is acceptable, and a 1500* read depth is suitable for detecting. Coverage is the average read depth of a specific region of DNA. Its aim is to have no missing area of target DNA.

Next-Generation Sequencing (NGS) is a powerful tool used for diagnosing rare diseases such as cancer, as well as for providing treatment guidance and conducting research in areas such as ecology, botany, and medical sciences. With NGS, both RNA and DNA can be sequenced, and it is possible to sequence the entire genome or transcriptome. Non-coding RNA, exome, cfDNA, methylation, and protein binding sites can also be sequenced using NGS.