High Throughput Sequencing: Difference between revisions
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High Throughput Sequencing is a technique used to sequence [[DNA|DNA]] or [[CDNA|cDNA]] via fragmenting the genetic information randomly. RNA can also be sequenced but involves additional steps to those shown below. Firstly [[MRNA|mRNA]] is hydrolysed by magnesium [[Catalysts|catalysed]] reaction to give fragments and then the sequences are randomly primed by [[Reverse transcription|reverse transcription]]. (Mardis | High Throughput Sequencing (also known as Illumina Solexa [[DNA|DNA]] sequencing) is a technique used to sequence [[DNA|DNA]] or [[CDNA|cDNA]] via fragmenting the genetic information randomly. RNA can also be sequenced but involves additional steps to those shown below. Firstly [[MRNA|mRNA]] is hydrolysed by [[Magnesium|magnesium]] [[Catalysts|catalysed]] reaction to give fragments and then the sequences are randomly primed by [[Reverse transcription|reverse transcription]] <ref>Mardis ER. (2008), "Next-generation DNA sequencing methods", Annu Rev Genomics Hum Genet.,9:387-402</ref><ref>Mardis ER (2008) Next-generation DNA sequencing methods.Annu Rev Genomics Hum Genet. 9:387-402</ref>.<br> | ||
'''The Process:''' | |||
===== Step 1 ===== | ===== Step 1 ===== | ||
Attach adapters to either end of the fragmented [[DsDNA|dsDNA]]. Two adapters are added separately so both ends of the [[DNA|dsDNA]] have an adapter present. | Double stranded DNA (dsDNA) is randomly fragmented to blund ended pieces. Attach adapters to either end of the fragmented [[DsDNA|dsDNA]] via blunt end ligation. Two adapters are added separately so both ends of the [[DNA|dsDNA]] have an adapter present. <br> | ||
<br> | ===== Step 2<br> ===== | ||
Denature the [[DNA|dsDNA]] to [[SsDNA|ssDNA]] and wash over flow cell channels. The adapters will enable binding to the dense lawn of [[Primers|primers]] on the flow cell.<br> | |||
Denature the [[DNA|dsDNA]] to [[SsDNA|ssDNA]] and wash over flow cell channels. The adapters will enable binding to the dense lawn of [[Primers|primers]] on the flow cell. | |||
<br> | |||
===== Step 3 ===== | ===== Step 3 ===== | ||
Bridge Amplification- | Bridge Amplification- The free end of the fragment bends and hybridises to a separate complimentary [[Primers|primer]] on the flow cell forming a 'bridge'. Unlabelled [[Nucleotides|nucleotides]] and [[Enzyme|enzymes]] are then added to the flow cell to initiate amplification of the second strand of [[DNA|DNA]].<br> | ||
<br> | |||
===== Step 4 ===== | ===== Step 4 ===== | ||
Denature [[DNA|DNA]] back into single stranded fragments | Denature [[DNA|DNA]] back into single stranded fragments and the process is repeated to produce clusters of identical [[DNA|DNA]] around the original sequence.<br> | ||
'''Step 5''' | |||
First Chemistry Cycle- Add four reversible terminators, [[Primers|primers]] and [[DNA|DNA]] [[Polymerase|polymerase]] to the flow cell. Each cluster will show up as spot after laser excitation due to the added bases fluorescence. The flow cell image is captured and the identity of the bases is recorded to determine the base sequence of the DNA. The blocked 3' OH terminus and the flurophone from each base are then removed to allow another base to be added to the sequence. These steps are being carried out repetitively until the base sequence of the DNA is completely sequenced. | |||
=== References === | === References === | ||
<references /><br> | |||
Latest revision as of 13:14, 12 October 2014
High Throughput Sequencing (also known as Illumina Solexa DNA sequencing) is a technique used to sequence DNA or cDNA via fragmenting the genetic information randomly. RNA can also be sequenced but involves additional steps to those shown below. Firstly mRNA is hydrolysed by magnesium catalysed reaction to give fragments and then the sequences are randomly primed by reverse transcription [1][2].
The Process:
Step 1
Double stranded DNA (dsDNA) is randomly fragmented to blund ended pieces. Attach adapters to either end of the fragmented dsDNA via blunt end ligation. Two adapters are added separately so both ends of the dsDNA have an adapter present.
Step 2
Denature the dsDNA to ssDNA and wash over flow cell channels. The adapters will enable binding to the dense lawn of primers on the flow cell.
Step 3
Bridge Amplification- The free end of the fragment bends and hybridises to a separate complimentary primer on the flow cell forming a 'bridge'. Unlabelled nucleotides and enzymes are then added to the flow cell to initiate amplification of the second strand of DNA.
Step 4
Denature DNA back into single stranded fragments and the process is repeated to produce clusters of identical DNA around the original sequence.
Step 5
First Chemistry Cycle- Add four reversible terminators, primers and DNA polymerase to the flow cell. Each cluster will show up as spot after laser excitation due to the added bases fluorescence. The flow cell image is captured and the identity of the bases is recorded to determine the base sequence of the DNA. The blocked 3' OH terminus and the flurophone from each base are then removed to allow another base to be added to the sequence. These steps are being carried out repetitively until the base sequence of the DNA is completely sequenced.