454 GS FLX Titanium system which is capable of generating 700 megabase (Mb) of sequence in 700 bp reads.
MiSeq reagents enable up to 15 Gb of output with 25 million sequencing reads and 2 × 300 bp read lengths.
In the first case to seq a total of 700 million bases it does by seq 700 base pairs at a time in a run?
In the second case to seq 15 billion bases what is 25 million sequencing reads and 2x300 bp reads.
And does the algorithm align these 300bp length sequences together to generate whole sequences?
Good afternoon,
I can try to clarify your doubts.
First of all, both methods generate sequences of a given length based on the primers used. The 454 can generate a sequence of maximum 700bp while the Illumina MiSeq generates 300bp reads. If your primers are less than 600bp far from each other, you will be able to overlap the two 300bp sequences into a single sequence (~600bp).
You will be able to merge the two 300bp sequences into a single one during your data analysis (this process is usually called "join paired ends").
A "read" is a single sequence generated by the MiSeq.
Targeting a single gene (for example 16S) from a pool of many different DNAs would result with the record of many different 16S sequences (reads) probably belonging to many different samples from the pool. Therefore, you could end up with 25 million reads, each of 600 bp. So the total number of bases (nucleotides) sequenced would be (25,000,000 x 600) = 15,000,000,000.
Hope this helps!
To generate 700mb seq optimal primer length would be 18-25 bp only right? same is the case with both 454 GS FLX and MiSeq? or only MiSeq generate it as two 300mb seq.
In order to align them in to one seq we create two 300mb overlapping seq combine it as one whole 600mb seq.
A 'read' is a single seq after join two 300bp into one 600bp? correct me if I'm wrong.
When targeting a single gene the primers will be specific to that single gene why would it generate 25 million reads? okay u mean from many DNA molecules harboring the same gene in the sample? how did they determine it as 25 million it could be less or more than that?
What if I deal with a new organism how do we seq its whole genome when the seq is not available on pubmed. Using random hexa primers or how do they do it these days? eg. nanopore offers real time DNA seq with out amplification or extraction?
"To generate 700mb seq optimal primer length would be 18-25 bp only right? same is the case with both 454 GS FLX and MiSeq? or only MiSeq generate it as two 300mb seq.
In order to align them in to one seq we create two 300mb overlapping seq combine it as one whole 600mb seq.
A 'read' is a single seq after join two 300bp into one 600bp? correct me if I'm wrong."
Correct, you have to generate a "forward" read and a "reverse" read that need to overlap. The combination of the two will give you a consensus read used downstream for the data analysis.
Just to be sure, we are talking of "base pairs" (bp), so each forward and reverse sequence needs to be maximum 300 nucleotides (for MiSeq) if you want them to overlap.
When targeting a single gene the primers will be specific to that single gene why would it generate 25 million reads? okay u mean from many DNA molecules harboring the same gene in the sample? how did they determine it as 25 million it could be less or more than that?
For example, if you have DNA extracted from a full insect, targeting the 16S rRNA you will be obtaining all the bacterial DNAs from the insect's guts and the insects symbionts. This can amount to hundreds and hundreds of different bacterial taxa. And each taxon can obviously be present in a multitude of individual bacteria. So, yeah, the numbers can increase pretty quickly!
However, I am not sure how they got that exact estimate (25 million reads). It may be a technical limitation instead of a biological one. Which means that probably MiSeq is not usually capable of generate more results. I am not sure about it, though.
What if I deal with a new organism how do we seq its whole genome when the seq is not available on pubmed. Using random hexa primers or how do they do it these days? eg. nanopore offers real time DNA seq with out amplification or extraction?
Well, if you want to sequence a whole genome you probably prefer a "long reads" sequencer (e.g. PacBio, Nanopore) instead of a "short reads" one (e.g. MiSeq). Or you can use a short reads sequencer in addition to the long reads one, too. These long reads sequencers can indeed generate very long sequences (more than 100Kb) that you will have to assemble into contigs until (hopefully) you will obtain a continuous sequence of each chromosome. But that's a whole different world, especially for the data analysis, as compared to short reads sequencing.
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