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general:bioseqanalysis:hifireads [2021/10/21 11:06] – yschoeneberggeneral:bioseqanalysis:hifireads [2021/10/21 11:12] (current) – yschoeneberg
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-The idea behind HIFI-reads is fairly simple. An adapter is added to the DNA-molecules, which circularizes them. This way it is possible for the polymerase to loop around the DNA-molecule several times, each time generating an error prone, "normal" long read, so called sub reads. Because the adapters are also sequenced one can easily tell where a new subread starts and where it ends. This way the subreads of the two DNA-strands can be compared and a consensus sequence for each read can be built. This way random sequencing errors cancel each other out, resulting in high accuracy long reads (Fig. {{ref>hifi-scheme}}). However, this technology is still quite expensive, since a lot of sequencing data is required. For example the approx. 9Gb of HIFI data were generated from approx. 560Gb of raw data.+The idea behind HIFI-reads is fairly simple. An adapter is added to the DNA-molecules, which circularizes them. This way it is possible for the polymerase to loop around the DNA-molecule several times, each time generating an error prone, "normal" long read, so called sub-reads. Because the adapters are also sequenced one can easily tell where a new subread starts and where it ends. This way the subreads of the two DNA-strands can be compared and a consensus sequence for each read can be built. This way random sequencing errors cancel each other out, resulting in high accuracy long reads (Fig. {{ref>hifi-scheme}}). However, this technology is still quite expensive, since a lot of sequencing data is required. For example the approx. 9Gb of HIFI data used in the course were generated from approx. 560Gb of raw data.
 <figure hifi-scheme> <figure hifi-scheme>
 {{ studentarea:ecoevo:2020:yannis_schoeneberg:hifi-reads_highly-accurate-long-reads-1.png?800 |}} {{ studentarea:ecoevo:2020:yannis_schoeneberg:hifi-reads_highly-accurate-long-reads-1.png?800 |}}
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-The high accuracy of HIFI-reads is reached by first circularizing the DNA using adapters and then sequencing the same DNA-molecule several times. Using the adapters one can easily tell where one so called subread starts and where it ends in the large polymerase read. This information can be used to build a circular consensus sequence, which has a sequencing error in the range of what is seen for the high quality Illumina reads (<0.1%).+The high accuracy of HIFI-reads is reached by first circularizing the DNA using adapters and then sequencing the same DNA-molecule several times. Using the adapters one can easily tell where one so called subread starts and where it ends in the large polymerase read. These subreads can be compared to each other and used to build a consensus sequence, in which random sequencing errors cancel each other out. This Circular Concensus Sequence (CCS) has a sequencing error in the range of what is seen for the high quality Illumina reads (<0.1%).
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