Re: [DIYbio] Geneome Sequencing Costs

That price is somewhat misleading. The way genome sequencing works is
you break the DNA into many small pieces and sequence the small
fragments. To be able to assemble all these small reads into a large,
contiguous chromosome the reads need to be overlapping. In fact, you
need a lot of overlap. If you are sequencing a genome for the first
time (de novo) then you probably need 100x coverage to be able to get
a good assembly. That means for every base pair in your genome you
need at least 100 reads to cover that spot. If you are re-sequencing
an already sequenced genome the necessary coverage goes down, but you
still need more than 1x coverage for a number of different reasons,
such as error minimization.

So if you are de novo sequencing something with a 37Mbp genome, you
will probably need at least 3.7Gbp worth of sequencing.

A couple of other complicating factors...

You can't just give your sample to a sequencing company and say, "give
me 1 million bases worth of sequencing." Assuming you are using an
Illumina HiSeq machine, your sample is going to be loaded into a thing
called a flow cell. That flow cell will give you a certain quantity
of base pairs regardless of how much you paid. That quantity depends
on how well your sample was prepared, how long the reads are, the
phase of the moon etc. So the cost is fixed per flow cell regardless
of how many of those reads you actually needed to get good coverage of
your genome.

Also, you have to factor in the cost of sample preparation. A lot of
work goes into preparing a sample for sequencing and the company will
probably charge you for it unless they have already factored that in
to the price. If you do a DNA extraction/purification (which costs
money) and send it off for sequencing, the company may determine it's
not pure enough or the DNA is too fragmented and you will probably get
charged for the assays they ran even though you got zero data.

Additionally, assuming you are de novo sequencing, after your first
run you go to assemble all those reads into a full chromosome. You
will probably find that you can't get them to assemble into a single
contiguous chromosome. You get a bunch of fragments which vary in
size from kilobases to megabases, called "contigs." How do you fill
in the gaps? Assuming the gaps are caused by regions of long repeats,
you can try to do some very long read sequencing, like 454 or PacBio
to bridge the gaps. If you think the gaps are caused by regions of
very high GC content, you can try to do some more Illumina sequencing
with an alternative kit that favors GC, or at least doesn't bias
towards regions of 40-60% GC. If you still can't fill in the gaps you
could try to scaffold your contigs against the genome of a related
species which already has a finished genome. This will give you a way
to order your contigs and estimate the size of the gaps so you can try
to use PCR to amplify the gaps. If you can clone the gap into a
plasmid or BAC then you can start Sanger sequencing on the gap DNA.
All of this costs more more money.

The upshot is that (1) it's impossible to determine upfront how much
it will cost to get a complete of genome of X megabases, and (2) you
can't sequence your mushroom for $37, unfortunately.

Sorry for the wall of text :)

-cory

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