The regular way to sequence the whole genome of SARS-CoV-2 is to use a PCR-based protocol like ARCTIC or MIDNIGHT, which actually don't even cover the whole genome because they're missing fragments of about 30-50 bases from the very beginning and end of the genome. For example in the third version of the ARCTIC protocol, the genome is co…
The regular way to sequence the whole genome of SARS-CoV-2 is to use a PCR-based protocol like ARCTIC or MIDNIGHT, which actually don't even cover the whole genome because they're missing fragments of about 30-50 bases from the very beginning and end of the genome. For example in the third version of the ARCTIC protocol, the genome is covered by 98 overlapping PCR amplicons with an average length of about 400 bases: https://github.com/artic-network/artic-ncov2019/blob/master/primer_schemes/nCoV-2019/V3/nCoV-2019.bed. But the first forward primer starts at position 30 of Wuhan-Hu-1 and the last reverse primer ends at position 29866, so the first 29 bases and last 66 bases of Wuhan-Hu-1 are not covered by any amplicon.
The protocols have to be updated every now and then because there's amplicon dropoff, because a part of the genome has mutated so it fails to be amplified by PCR. So because of that the protocols also have alternative primers for a couple of locations that have mutated too much so they have amplicon dropoff.
In the paper by Fan Wu et al., the Wuhan-Hu-1 reference genome was initially determined by doing metagenomic RNA-seq with random priming and then by doing de-novo assembly with MEGAHIT. But there's probably not too many sequences of SARS-CoV-2 submitted to GISAID that were sequenced by doing metagenomic sequencing or that were assembled with a de-novo assembler like MEGAHIT.
PCR-based sequencing is cheaper than metagenoming or total RNA sequencing, because you don't have to sequence as much genetic material to get high coverage for the viral genome.
The regular way to assemble the reads is to align the reads against a reference genome with a short read aligner like minimap2 or BWA. De-novo assemblers like MEGAHIT are slower, and they sometimes end up missing a short piece from either end of the genome, or they fail to produce a single complete contig for the entire genome. And they sometimes make an error where either extreme of the viral contig includes a short piece of host ribosomal RNA or a copy of a wrong part of the viral sequence.
And also variant calling workflows are meant to be used with a BAM/SAM file of raw reads that were aligned against a reference, so they can't be used with a FASTA file produced by MEGAHIT which contains the whole genome in a single piece but which is missing base quality scores.
I wasn't talking about PCR testing but about a method used in genetic sequencing where PCR is used to amplify overlapping regions of the genome of a virus. The purpose of PCR in that case is to reduce sequencing costs by making more copies of the viral genome, so that you can get many reads which cover the genome without having to sequence gigabytes of genetic material.
Kary Mullis wrote: "I will not try to convince anyone that PCR can be used successfully to specifically make multiple copies of any nucleic acid sequence that can be uniquely defined by two 'primer target sequences' comprising the termini of the sequence of interest. The veracity of this no longer has anything to do with me. I think this has been confirmed by a huge number of laboratories around the world. The rapid spread of this simple technology would not have occurred had it been ineffectual or flawed in any persistent way." (http://aras.ab.ca/articles/legal/McDonald-Mullis.html)
basic premis is perfidous therefore all that fol;lows are the perfidous fruits kerry mullis states that its not a test its a method of replication not identification
the creator of the pcr kerryu mullins said it cannot be used as a test ,,, pcr can only replicate ... and you choose to ignore this truth by the inventor of pcr , thats equats to tremendous dizzing specular ego mania or tremendopus perfidy
Hey Alex, you should IGNORE "henjin", but you really would be well advised to stop citing Kary Mullis! He was NOT a hero to our old "AIDS" dissident movement! This quote is NOT what it seems to be! Dr. Kary Mullis' EXACT words were: "It (PCR) doesn't tell you that you're sick, and it doesn't tell you that the thing you ended up with really was gonna hurt you, or anything like that"...!! I was THERE when Mullis uttered these words back in 1997. I helped to produce the seminar where Mullis said this! Mullis BELIEVED in viruses, and he DEFINITELY believed that his PCR could INDEED detect viruses. Mullis was PART of the PROBLEM! Please see:
friend thanks for trying to put me on the right truth track,,,i read all the links 6they are all commtaries on whatkary alledgedly said and quite out of context . i think ... i learned to read from the source, in this case vid footage although fabrication ommisions abound ... i couldnt find an unedited uncensored original that i saw at the begining of thisw sad event...yes the pcr could detect viruses provided the virus has been identified isolated , which is quintessental , then the pcr can work otherwise as kary says in the vid , you dont know what genes your looking at ... finally ,,,, yandex came up w3ith the vid , thats the link i send as i remember the vid... your links are well perfidous by omission,,,please no offence intended by me ... just truth... good God bless everyone ... happy trails yippie ya yea...
Alex I'm really sorry that you continue to be confused even after I supplied links that should provide you with clarification about the "role" that Kary Mullis actually played in our dear old "AIDS" dissident movement. However, in the VERY SHORT span of time that you took to post your reply, it's not even possible for you to have truly absorbed everything at all of the links I provided . As such, I don't believe there is anything else I can do at this point to try to enlighten you any further. God bless YOU too, and happy trails to you, too friend...!
thank you for your good intent,[ i can do simple speed reading ,, as far as kary ...im not interested in personalities in this subject... just pcr facts... pcr can detect and identify any gene provided its been isolated and been proven to be causitive of illness... kovid hasnt been isolated ,,, its genetics were created on with computer etc ... i wish you all the best
only if its been isolated and proven to cause illness ie a referance gene identified then pcr can multiply that gene ... if there is no isolated proven causitive gene then pcr has no reference , and the scientist dont know what gene they are looking for what has been multiplied by pcr ,, because there are multitude of various genes ... everything rests on isolation of the genetic causative... and kovvidd has not been isolated...
In the patent for PCR that was issued in 1986 to Kary Mullis et al., they wrote that one application for PCR would be to diagnose the presence of pathogenic microorganisms including viruses. [https://patents.google.com/patent/US4683195] Mullis was also the last author of a patent from 1989 titled "Detection of viruses by amplification and hybridization", where they specifically wrote that PCR can be used to detect HIV, and they wrote that HIV has been sequenced and that there are isolates of HIV available. [https://patents.google.com/patent/US5176995A] Kary Mullis was additionally one of the authors of a paper published in 1987 titled "Identification of Human Immunodeficiency Virus Sequences by Using In Vitro Enzymatic Amplification and Oligomer Cleavage Detection". [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC254157/pdf/jvirol00096-0400.pdf]
When Kary Mullis said that "quantitative PCR is an oxymoron", I believe he meant that PCR was not an accurate way to quantify viral load, and not that PCR was not an accurate way to determine whether a sample contains a virus or not. John Lauritsen wrote: "With regard to the viral load tests, which attempt to use PCR for counting viruses, Mullis has stated: 'Quantitative PCR is an oxymoron.' PCR is intended to identify substances qualitatively, but by its very nature is unsuited for estimating numbers." [https://www.virusmyth.com/aids/hiv/jlprotease.htm]
In an interview with Gary Null, Mullis also said: "PCR came along right about the same time that HIV did. And it was in that [unintelligible word] that people started looking with PCR for HIV. That was the only way to see it, except for culture. Which was a long protracted procedure, which a lot of times didn't turn right. [...] The culture - the whole method - cell biology is a bunch of magic half of the time. And people who say that they can do quantitative estimations of HIV from culture, they're just - they're fooling themselves." [https://www.bitchute.com/video/8SjzUDxBZL9t/, time 9:05] But by "quantitative estimations", I think Mullis was again talking about estimating viral load.
HIV is typically detected using antigen or antibody tests and not PCR, but the main use of PCR tests in the case of HIV was traditionally to measure viral load. PCR tests for HIV are commonly called "NAT tests" or nucleic acid tests, and they are further divided into quantitative and non-quantitative NAT tests depending on whether their aim is to measure viral load, even though some NAT tests for HIV also use transcription-mediated amplification instead of PCR. So the typical use of PCR tests for HIV is different from the typical use of PCR tests for COVID.
The CDC's website says that "Most rapid tests and the only HIV self-test approved by the U.S. Food and Drug Administration (FDA) are antibody tests." [https://www.cdc.gov/hiv/basics/hiv-testing/test-types.html] And the CDC's website also says that a NAT test "can tell if a person has HIV or how much virus is present in the blood (HIV viral load test)" and that a NAT test "should be considered for people who have had a recent exposure or a possible exposure and have early symptoms of HIV and who have tested negative with an antibody or antigen/antibody test" (so basically a NAT test is used as a fallback for antibody and antigen tests).
In the year 2006 in Australia, there was a court case where someone was convicted of three counts of endangering life after he had unprotected sex with three women without telling them that he was HIV positive. [https://web.archive.org/web/20070709210442/http://garlan.org/Cases/Parenzee/2007-SASC-143-Parenzee.pdf] He appealed by stating that HIV has not been proven to exist, and testimony in his favor was provided by Eleni Papadopolous-Eleopulos and Valendar Turner from the Perth Group. After Mullis's comments about PCR were brought up the Perth Group, Mullis was sent an e-mail which said: "I am assisting the prosecution in an Appeal to the Supreme court in South Australia about a conviction for criminal transmission of HIV. The basis for that Appeal is that HIV does not exist and that the PCR technology is flawed. So in effect the technical basis for identification of virus is on trial. The group of denialists giving evidence are people from Perth who quote you as indicating that PCR technology is erroneous and misleading. Can I ask you to comment on this statement." [http://aras.ab.ca/articles/legal/McDonald-Mullis.html] But Mullis responded by writing: "I will not try to convince anyone that PCR can be used successfully to specifically make multiple copies of any nucleic acid sequence that can be uniquely defined by two 'primer target sequences' comprising the termini of the sequence of interest. The veracity of this no longer has anything to do with me. I think this has been confirmed by a huge number of laboratories around the world. The rapid spread of this simple technology would not have occurred had it been ineffectual or flawed in any persistent way." And Mullis also wrote that "the AIDS/HIV issue is what is not settled scientifically, not the effectiveness of PCR".
I just want to applaud you. Even though we seem to disagree on the conclusions one can draw from these studies, you are one of the only people I have ever met who has actually read and familarized yourself with the historical criticism of virus theory and then discussed it. So, even though we seem to disagree on whether the theory is valid or not, I do thank you for joining the discussion and doing so in a contemplative way!
Whats written in a patent is a claim. It doesn't necessarily have to be capable of being "reduced to practise".
I suspect, because I've done it myself, is to disclose a use OTHERS might want to patent. You do this because then you've "prior arted them".
They cannot secure a patent unless something is NEW, INVENTIVE and USEFUL.
If its disclosed already, you cannot get a strong patent on anything using that which is in the public domain already, because your invention lacks novelty.
The regular way to sequence the whole genome of SARS-CoV-2 is to use a PCR-based protocol like ARCTIC or MIDNIGHT, which actually don't even cover the whole genome because they're missing fragments of about 30-50 bases from the very beginning and end of the genome. For example in the third version of the ARCTIC protocol, the genome is covered by 98 overlapping PCR amplicons with an average length of about 400 bases: https://github.com/artic-network/artic-ncov2019/blob/master/primer_schemes/nCoV-2019/V3/nCoV-2019.bed. But the first forward primer starts at position 30 of Wuhan-Hu-1 and the last reverse primer ends at position 29866, so the first 29 bases and last 66 bases of Wuhan-Hu-1 are not covered by any amplicon.
The protocols have to be updated every now and then because there's amplicon dropoff, because a part of the genome has mutated so it fails to be amplified by PCR. So because of that the protocols also have alternative primers for a couple of locations that have mutated too much so they have amplicon dropoff.
In the paper by Fan Wu et al., the Wuhan-Hu-1 reference genome was initially determined by doing metagenomic RNA-seq with random priming and then by doing de-novo assembly with MEGAHIT. But there's probably not too many sequences of SARS-CoV-2 submitted to GISAID that were sequenced by doing metagenomic sequencing or that were assembled with a de-novo assembler like MEGAHIT.
PCR-based sequencing is cheaper than metagenoming or total RNA sequencing, because you don't have to sequence as much genetic material to get high coverage for the viral genome.
The regular way to assemble the reads is to align the reads against a reference genome with a short read aligner like minimap2 or BWA. De-novo assemblers like MEGAHIT are slower, and they sometimes end up missing a short piece from either end of the genome, or they fail to produce a single complete contig for the entire genome. And they sometimes make an error where either extreme of the viral contig includes a short piece of host ribosomal RNA or a copy of a wrong part of the viral sequence.
And also variant calling workflows are meant to be used with a BAM/SAM file of raw reads that were aligned against a reference, so they can't be used with a FASTA file produced by MEGAHIT which contains the whole genome in a single piece but which is missing base quality scores.
However besides the metagenomic sequencing reads published by Fan Wu et al., there are also metagenomic sequencing reads of other early samples that are available from the NCBI's Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra/?term=SRR11092064). And there are metagenomic runs of environmental samples from the Huanan Seafood Market that contain enough reads of SARS-CoV-2 that they can be used to assemble the genome (https://github.com/jbloom/Huanan_market_samples/blob/main/results/aggregated_counts/sars2_aligned_by_run.csv). And there are metagenomic sequencing runs of other SARS-like coronaviruses that didn't use targeted enrichment so they didn't specifically amplify SARS-like viruses (https://www.ncbi.nlm.nih.gov/sra/?term=SRR11085797).
https://wickedtruths.org/en/kary-mullis/ ... VID KARY STATES THAT PCR IS NOT A TEST NOR CAN BE AN ACCURATE ONE
I wasn't talking about PCR testing but about a method used in genetic sequencing where PCR is used to amplify overlapping regions of the genome of a virus. The purpose of PCR in that case is to reduce sequencing costs by making more copies of the viral genome, so that you can get many reads which cover the genome without having to sequence gigabytes of genetic material.
Kary Mullis wrote: "I will not try to convince anyone that PCR can be used successfully to specifically make multiple copies of any nucleic acid sequence that can be uniquely defined by two 'primer target sequences' comprising the termini of the sequence of interest. The veracity of this no longer has anything to do with me. I think this has been confirmed by a huge number of laboratories around the world. The rapid spread of this simple technology would not have occurred had it been ineffectual or flawed in any persistent way." (http://aras.ab.ca/articles/legal/McDonald-Mullis.html)
basic premis is perfidous therefore all that fol;lows are the perfidous fruits kerry mullis states that its not a test its a method of replication not identification
the creator of the pcr kerryu mullins said it cannot be used as a test ,,, pcr can only replicate ... and you choose to ignore this truth by the inventor of pcr , thats equats to tremendous dizzing specular ego mania or tremendopus perfidy
Hey Alex, you should IGNORE "henjin", but you really would be well advised to stop citing Kary Mullis! He was NOT a hero to our old "AIDS" dissident movement! This quote is NOT what it seems to be! Dr. Kary Mullis' EXACT words were: "It (PCR) doesn't tell you that you're sick, and it doesn't tell you that the thing you ended up with really was gonna hurt you, or anything like that"...!! I was THERE when Mullis uttered these words back in 1997. I helped to produce the seminar where Mullis said this! Mullis BELIEVED in viruses, and he DEFINITELY believed that his PCR could INDEED detect viruses. Mullis was PART of the PROBLEM! Please see:
1. https://docs.google.com/document/d/1fbpYott1Mdw_BuuiE6_rjO_7Xr5Z_PdoxtK3fNZI9Gc
2. https://longtimedissident.substack.com/p/the-mullis-mirage
3. https://longtimedissident.substack.com/p/was-mullis-more-machiavellian-than
friend thanks for trying to put me on the right truth track,,,i read all the links 6they are all commtaries on whatkary alledgedly said and quite out of context . i think ... i learned to read from the source, in this case vid footage although fabrication ommisions abound ... i couldnt find an unedited uncensored original that i saw at the begining of thisw sad event...yes the pcr could detect viruses provided the virus has been identified isolated , which is quintessental , then the pcr can work otherwise as kary says in the vid , you dont know what genes your looking at ... finally ,,,, yandex came up w3ith the vid , thats the link i send as i remember the vid... your links are well perfidous by omission,,,please no offence intended by me ... just truth... good God bless everyone ... happy trails yippie ya yea...
Alex I'm really sorry that you continue to be confused even after I supplied links that should provide you with clarification about the "role" that Kary Mullis actually played in our dear old "AIDS" dissident movement. However, in the VERY SHORT span of time that you took to post your reply, it's not even possible for you to have truly absorbed everything at all of the links I provided . As such, I don't believe there is anything else I can do at this point to try to enlighten you any further. God bless YOU too, and happy trails to you, too friend...!
thank you for your good intent,[ i can do simple speed reading ,, as far as kary ...im not interested in personalities in this subject... just pcr facts... pcr can detect and identify any gene provided its been isolated and been proven to be causitive of illness... kovid hasnt been isolated ,,, its genetics were created on with computer etc ... i wish you all the best
How do you know that PCR can detect and identify any gene?
only if its been isolated and proven to cause illness ie a referance gene identified then pcr can multiply that gene ... if there is no isolated proven causitive gene then pcr has no reference , and the scientist dont know what gene they are looking for what has been multiplied by pcr ,, because there are multitude of various genes ... everything rests on isolation of the genetic causative... and kovvidd has not been isolated...
Are you saying that genes can be isolated?
If yes, how do you know this?
https://duckduckgo.com/?t=ffab&q=gene+isolation&atb=v223-1&ia=web
I do not know why you posted some link.
Again. How do you know this?
Describe with your own words.
In the patent for PCR that was issued in 1986 to Kary Mullis et al., they wrote that one application for PCR would be to diagnose the presence of pathogenic microorganisms including viruses. [https://patents.google.com/patent/US4683195] Mullis was also the last author of a patent from 1989 titled "Detection of viruses by amplification and hybridization", where they specifically wrote that PCR can be used to detect HIV, and they wrote that HIV has been sequenced and that there are isolates of HIV available. [https://patents.google.com/patent/US5176995A] Kary Mullis was additionally one of the authors of a paper published in 1987 titled "Identification of Human Immunodeficiency Virus Sequences by Using In Vitro Enzymatic Amplification and Oligomer Cleavage Detection". [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC254157/pdf/jvirol00096-0400.pdf]
When Kary Mullis said that "quantitative PCR is an oxymoron", I believe he meant that PCR was not an accurate way to quantify viral load, and not that PCR was not an accurate way to determine whether a sample contains a virus or not. John Lauritsen wrote: "With regard to the viral load tests, which attempt to use PCR for counting viruses, Mullis has stated: 'Quantitative PCR is an oxymoron.' PCR is intended to identify substances qualitatively, but by its very nature is unsuited for estimating numbers." [https://www.virusmyth.com/aids/hiv/jlprotease.htm]
In an interview with Gary Null, Mullis also said: "PCR came along right about the same time that HIV did. And it was in that [unintelligible word] that people started looking with PCR for HIV. That was the only way to see it, except for culture. Which was a long protracted procedure, which a lot of times didn't turn right. [...] The culture - the whole method - cell biology is a bunch of magic half of the time. And people who say that they can do quantitative estimations of HIV from culture, they're just - they're fooling themselves." [https://www.bitchute.com/video/8SjzUDxBZL9t/, time 9:05] But by "quantitative estimations", I think Mullis was again talking about estimating viral load.
HIV is typically detected using antigen or antibody tests and not PCR, but the main use of PCR tests in the case of HIV was traditionally to measure viral load. PCR tests for HIV are commonly called "NAT tests" or nucleic acid tests, and they are further divided into quantitative and non-quantitative NAT tests depending on whether their aim is to measure viral load, even though some NAT tests for HIV also use transcription-mediated amplification instead of PCR. So the typical use of PCR tests for HIV is different from the typical use of PCR tests for COVID.
The CDC's website says that "Most rapid tests and the only HIV self-test approved by the U.S. Food and Drug Administration (FDA) are antibody tests." [https://www.cdc.gov/hiv/basics/hiv-testing/test-types.html] And the CDC's website also says that a NAT test "can tell if a person has HIV or how much virus is present in the blood (HIV viral load test)" and that a NAT test "should be considered for people who have had a recent exposure or a possible exposure and have early symptoms of HIV and who have tested negative with an antibody or antigen/antibody test" (so basically a NAT test is used as a fallback for antibody and antigen tests).
In the year 2006 in Australia, there was a court case where someone was convicted of three counts of endangering life after he had unprotected sex with three women without telling them that he was HIV positive. [https://web.archive.org/web/20070709210442/http://garlan.org/Cases/Parenzee/2007-SASC-143-Parenzee.pdf] He appealed by stating that HIV has not been proven to exist, and testimony in his favor was provided by Eleni Papadopolous-Eleopulos and Valendar Turner from the Perth Group. After Mullis's comments about PCR were brought up the Perth Group, Mullis was sent an e-mail which said: "I am assisting the prosecution in an Appeal to the Supreme court in South Australia about a conviction for criminal transmission of HIV. The basis for that Appeal is that HIV does not exist and that the PCR technology is flawed. So in effect the technical basis for identification of virus is on trial. The group of denialists giving evidence are people from Perth who quote you as indicating that PCR technology is erroneous and misleading. Can I ask you to comment on this statement." [http://aras.ab.ca/articles/legal/McDonald-Mullis.html] But Mullis responded by writing: "I will not try to convince anyone that PCR can be used successfully to specifically make multiple copies of any nucleic acid sequence that can be uniquely defined by two 'primer target sequences' comprising the termini of the sequence of interest. The veracity of this no longer has anything to do with me. I think this has been confirmed by a huge number of laboratories around the world. The rapid spread of this simple technology would not have occurred had it been ineffectual or flawed in any persistent way." And Mullis also wrote that "the AIDS/HIV issue is what is not settled scientifically, not the effectiveness of PCR".
I just want to applaud you. Even though we seem to disagree on the conclusions one can draw from these studies, you are one of the only people I have ever met who has actually read and familarized yourself with the historical criticism of virus theory and then discussed it. So, even though we seem to disagree on whether the theory is valid or not, I do thank you for joining the discussion and doing so in a contemplative way!
Whats written in a patent is a claim. It doesn't necessarily have to be capable of being "reduced to practise".
I suspect, because I've done it myself, is to disclose a use OTHERS might want to patent. You do this because then you've "prior arted them".
They cannot secure a patent unless something is NEW, INVENTIVE and USEFUL.
If its disclosed already, you cannot get a strong patent on anything using that which is in the public domain already, because your invention lacks novelty.
Good points.
(sorry but my "like" function is somehow disabled)