cost of sequencing(2)

from PFI

Posted: Sun Aug 05, 2007 6:50 am  Post subject: 

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Sequencing is very cheap and getting cheaper. There is no reason for not releasing the complete sequence of all 8 genomic segments for mulitple isolates from every patient.
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Pixie
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  Posted: Sun Aug 05, 2007 6:53 am  Post subject: 

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gs, that's an interesting chart. I did not know there was anything from as long ago as 1902 to sequence.

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gsgs

Joined: 08 Dec 2006
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  Posted: Sun Aug 05, 2007 7:31 am  Post subject: 

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any problem with sequencing old, 100 year-old samples then ?
We're still waiting for the sequences from the English diplomat
and the announced sequences from the 1918 first wave.

Is it all political or security concerns or do they
want it exclusively, wnt to be the first to publish a paper about it ?

here is the paper about the very short bird sequences
from 1916,1917:

http://jvi.asm.org/c...
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Monotreme
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  Posted: Sun Aug 05, 2007 11:07 am  Post subject: 

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gsgs, the problem with old viruses is not sequencing per se, its getting something to sequence. RNA is very easily degraded, ie, chopped up into small pieces. The longer the time from sample collection to turning the RNA to cDNA (which is much more stable), the worse the situation will be. Taubenberger et al have done a remarkable job of reassembling sequence from severely degraded samples.
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Pixie
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  Posted: Sun Aug 05, 2007 11:13 am  Post subject: 

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Just an intellectual exercise - what are the oldest samples we might have available to sequence? What kind of environment would they have had to be kept in to remain viable and possibly meet the criteria Monotreme laid out above?

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Monotreme
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  Posted: Sun Aug 05, 2007 11:20 am  Post subject: 

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Pixie, frozen in a lab is best. Frozen in a corpse has also worked.

Even back in 1918, pathology samples have been fixed in formalin and embedded in paraffin blocks for sectioning to make microscope slides. Some of these paraffin blocks still exist and have been used to obtain sequence.

Initial Genetic Characterization of the 1918 "Spanish" Influenza Virus
http://www.sciencema...

The problem with this approach is that there are probably few if any paraffin blocks from bird samples.
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gsgs

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  Posted: Sun Aug 05, 2007 11:29 am  Post subject: 

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maybe someone with flu, surprised in the Alpes by an avalanche
covered with tons of snow

or just some bird dying of flu in arctic area, then it snows,
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gsgs

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  Posted: Sun Aug 05, 2007 11:30 am  Post subject: 

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monotreme, that's what they did for the birds from 1917:
http://jvi.asm.org/c...
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gsgs

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  Posted: Sun Aug 05, 2007 11:31 am  Post subject: 

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hmm, do you think Taubenerger et.al have more
sequence - fragments, which they just only didn't upload
because genbank requires contiguous sequences ??

someone should ask...maybe SusanC

 

ask experts for their subjective
panflu death expectation values
and report the replies

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by: gs @ Sun Aug 05, 2007 at 16:57:43 PM UTC

first of all

you need to get that not everything has to do with cost.  ;-)  I'm not saying it isn't, but it's not always the limiting factor.  There have been huge amounts of money invested on sequencing in various institutions, the NIH being one of them.

When you are talking about sequencing from historical samples, what I understand from Taubenberger is that fragments from pathology samples are very small.  Secondly, it depends on the amount of viral RNA present at the time of death.  The 1918 pandemic was unique in the speed with which people died plus the massive amount of viral replication occurring. 

Relative to that, if you are trying to find fragments pre-1918, you are talking about seasonal flu.  There is much lower viral replication, plus people die later, often from other complications such as bacterial pneumonia.  ie the chance of getting autopsy samples from someone dying at the time of maximal and massive viral replication is quite low.

Even when you have the fragments, it's hard to determine what you are looking at because the fragments are so small.  It's a little like putting together a puzzle.  If the puzzle pieces are very small and you don't have all the pieces, it is a lot of work to put the puzzle together.



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...

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by: SusanC @ Sun Aug 05, 2007 at 17:11:54 PM UTC

[ Parent ]

give us the fragments

I'm expert for computer-puzzling ;-)

ask experts for their subjective
panflu death expectation values
and report the replies

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by: gs @ Sun Aug 05, 2007 at 17:57:29 PM UTC

[ Parent ]

if they can't do it

at Taubenberger's lab, I doubt that anyone can do it, at this point!  LOL

I'm not saying they can't do it.  It's just going to take a long time and a lot of effort to put them together.

There is keen competition in science.  If someone else can do it, they would have done it by now!  Access to the pathology samples is not restricted.  A lot of what they are working on come from hospital archives from London, which are not really secret or classified in any way.



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...

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by: SusanC @ Sun Aug 05, 2007 at 18:02:30 PM UTC

[ Parent ]

to give a different perspective

It's important to not just focus on individual sequences.  A lot of work is going on with sequencing of large numbers of samples from avian and human origins, with the Influenza Virus Genome Project .  The following chart gives you an idea of the rapid increase in number of sequences completed.

Many lessons can be learnt from looking at the big picture relationship between the different sequences in relation to their ecological environment eg host and ecological determinants of viral 'fitness', mechanisms for host switching, etc.  In order to understand better what any individual sequence means, we need to know a lot more about the underlying science.  Building up that knowledge is going to give us a lot more tools to understand the significance of individual changes.



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...

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by: SusanC @ Sun Aug 05, 2007 at 18:24:57 PM UTC

[ Parent ]

AT/CG-ratio

yes, I know. I have all those sequences.

for the moment
I just want the ratio of A,T vs. C,G  nucleotides,
that should also be possible with fragments.
It helps to determine, how human-like a virus is.

all
human:.5701,.5780,.5738,.5814,.5372,.5722,.5269,.5672
swine:.5615,.5743,.5679,.5875,.5409,.5766,.5242,.5642
avian:.5537,.5678,.5599,.5793,.5265,.5678,.5161,.5631

H1N1:
human:.5770,.5867,.5798,.5877,.5373,.5843,.5333,.5631
swine:.5656,.5769,.5747,.5945,.5409,.5780,.5270,.5652
avian:.5496,.5698,.5614,.5758,.5279,.5729,.5172,.5596

1918-virus:.5561,.5659,.5620,.5802,.5310,.5751,.5152,.5465

looks pretty much avian in all segments, except NA and maybe PB2.

Would be interesting, how other viruses from that time score
to look for possible reassortments.

He can just give the ratios from the fragments without
having to disclose them !

ask experts for their subjective
panflu death expectation values
and report the replies

---

by: gs @ Sun Aug 05, 2007 at 19:20:04 PM UTC

[ Parent ]

reference from JKT

He says he doesn't have anything to add at this point, but would refer you to this:

Rabadan R, Levine AJ, Robins H. Comparison of avian and human influenza A viruses reveals a mutational bias on the viral genomes. J Virol. 2006 Dec;80(23):11887-91. Epub 2006 Sep 20.



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...

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by: SusanC @ Tue Aug 07, 2007 at 18:24:10 PM UTC

[ Parent ]

Rabadan et.al

yes, we had discussed this last year.
Even if fragments can't be joined, they could still be
useful for AT/CG analysis.

so please give us the fragments from before 1918 !

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---

by: gs @ Thu Aug 09, 2007 at 10:11:31 AM UTC

[ Parent ]

what do you mean?

As far as I understand, whatever they have sequenced are all at NCBI.



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...

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by: SusanC @ Thu Aug 09, 2007 at 10:19:53 AM UTC

[ Parent ]

sequencing old samples

as I understood...
sequencing is easy and cheap.
The reason why we don't get the old sequences,
is because the genes are broken and they only get
small subsequences with PCR.
So, for one such uploaded segment of 100 nucleotides,
I assume they have dozends of shorter ones, which
they can't join to larger ones.
How else can it be that we get short subsequences
but not long ones ?

ask experts for their subjective
panflu death expectation values
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---

by: gs @ Thu Aug 09, 2007 at 14:52:42 PM UTC

[ Parent ]

just the result ?

he needn't even publish the small fragments which he
presumably has.
Run a (A+T)/(C+G) count through all the fragments
from one segment and just only give us that number.

ask experts for their subjective
panflu death expectation values
and report the replies

---

by: gs @ Mon Aug 13, 2007 at 06:41:47 AM UTC

[ Parent ]

follow up paper

http://www.plospathogens.org/a...

they found the pattern in other human viruses too.
Also a lower frequency of xxCGxx in dicodons
of human viruses.
Improves our reliability to identify human viruses.
Some human or swine flu-virus-fragments from before 1918
would be interesting.

The explanation is unclear, but the same pattern
for xxCGxx - frequency appears in the host

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---

by: gs @ Thu Jun 12, 2008 at 14:29:01 PM UTC

[ Parent ]

date errors

(the 1909,1911 sequences, are they date-errors ?)

Yes, I asked JKT and he said the 1909 and 1911 were all actually 2003 samples.  He has already notified NCBI of this error.

Basically, the only pre-1918 samples are 1916 & 1917 smithsonian samples,
AY220475 151 Avian 5 (NP)  USA 1916 Influenza A virus (A/cinnamon teal/Utah/1/1916)
  AY095226 166 Avian 4 (HA) H1 USA 1917 Influenza A virus (A/Brant Goose/1/1917(H1N?))
  AY095227 288 Avian 4 (HA) H1 USA 1917 Influenza A virus (A/Brant Goose/1/1917(H1N?))
  AY095228 151 Avian 5 (NP) H1 USA 1917 Influenza A virus (A/Brant Goose/1/1917(H1N?))

and the 1902 HPAI H7N7 from Italy, which was isolated as a "fowl plague virus".  It wasn't known until 1950's that FPV was HPAI.  The problem is these old strains had to be continually passaged before deep freezes were invented, so they may have acquired lab-adapted changes.



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...

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by: SusanC @ Tue Aug 07, 2007 at 18:22:05 PM UTC

1902

from the 1902 Brescia sample the full genome is available.
But it's almost identical to the 1927 Dobson virus.

Can it be ?

ask experts for their subjective
panflu death expectation values
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---

by: gs @ Thu Aug 09, 2007 at 10:14:05 AM UTC

[ Parent ]

Hard to say

cos of the repeated passage, they may have, as he said, acquired lab-adapted changes.



All 'safety concerns' are hypothetical.  If not, they'd be called side effects...

---

by: SusanC @ Thu Aug 09, 2007 at 10:20:37 AM UTC

[ Parent ]