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Is there a way to get just the DNA you're interested in sequencing in the first place? Eliminating the sequences that look out of place afterwards does not seem

Is there a way to get just the DNA you're interested in sequencing in the first place? Eliminating the sequences that look out of place afterwards does not seem like a good strategy.

GFK_of_xmaspast 11y ago
Supposing there was, why wouldn't they have done that?
- stefantalpalaru 11y ago
  Cost.
noname123 11y ago
Sequencing is typically done in "whole-genome shotgun" fashion; or imagine, if you shred a 100pg document (lyse and extract DNA, Illumina sequencing) into 100 lettered fragments, and have gathered all of the fragments of 1000 copies of this shredded document, some of them are regular words in forward orientation and some of them have every letter reversed (reverse strand since DNA is complementary double-stranded); The fragments given the nature of the shredder are all shredded slightly at different places and they are all mixed up - so you have to piece together like a jigsaw puzzle into a single copy (genome assembly). However suppose there are lots of palindromic repeats in the document like the phrase "stressed" repeated 100 times such that they exceed your individual 100 lettered fragment, or is it the palindrome dopleganger "desserts" repeated 100 times? Or a mix of "stressed desserts" repeated in different ways 100 times? And also what of the phrases before and after "stressed/desserts," how do you place them since you are not sure about the order/orientation of this repeat? This is the challenge of genome assembly. Now having more copies of the shredded document helps but some repeats are very nasty and aligning them in the assembly is not statistically tractable. And also they don't necessarily encode for relevant genes so they are often filtered out. Furthermore, they are arguably the regions of the DNA that is the most prone to change, since you can think of them as the "filler text" of the document; if an important part of the document is unintelligible, then this document wouldn't survive nature's QC process of sexual selection. However, unintelligible parts of the document don't encode important genes, so they evolve very quickly because they have a higher tolerance of typo's and mutations. So in an population pool that people use to assemble and come up with an consensus genome (keep in mind the population pool will have different gene variation), so it may turn out at a particular base pair position in the document, there is so much variation that the genome assembler can't conclusively come up with an consensus sequence or a reasonable distribution of base pair (A, T, C, G).
ende 11y ago
That would be ideal. Unfortunately sequencing is imperfect and there are many points throughout the process where error can be introduced. The easiest source of error is simple contamination. It happens even to the best of bench biologists.
88e282102ae2e5b 11y ago
Not really. If you want tardigrade DNA, you have to break open their cells one way or another. In the pre-print referenced in the article, they basically just froze the tardigrades to -80°C and mechanically smashed them. The problem is that bacteria are pretty fragile. So if you have a tardigrade that has some bacteria sitting on it (or the tardigrade just ate some), whatever process you use to extract the tardigrade DNA is also going to extract the bacterial DNA. Since this is done in solution, the bacterial and tardigrade DNA will be irrevocably mixed. While they do wash and filter the tardigrades extensively and also starve them for several days before extracting their DNA, that's still not enough to completely remove all bacteria.

Is there a way to get just the DNA you're interested in sequencing in the first place? Eliminating the sequences that look out of place afterwards does not seem like a good strategy.

GFK_of_xmaspast 11y ago

Supposing there was, why wouldn't they have done that?

stefantalpalaru 11y ago
Cost.

noname123 11y ago

Sequencing is typically done in "whole-genome shotgun" fashion; or imagine, if you shred a 100pg document (lyse and extract DNA, Illumina sequencing) into 100 lettered fragments, and have gathered all of the fragments of 1000 copies of this shredded document, some of them are regular words in forward orientation and some of them have every letter reversed (reverse strand since DNA is complementary double-stranded); The fragments given the nature of the shredder are all shredded slightly at different places and they are all mixed up - so you have to piece together like a jigsaw puzzle into a single copy (genome assembly). However suppose there are lots of palindromic repeats in the document like the phrase "stressed" repeated 100 times such that they exceed your individual 100 lettered fragment, or is it the palindrome dopleganger "desserts" repeated 100 times? Or a mix of "stressed desserts" repeated in different ways 100 times? And also what of the phrases before and after "stressed/desserts," how do you place them since you are not sure about the order/orientation of this repeat? This is the challenge of genome assembly. Now having more copies of the shredded document helps but some repeats are very nasty and aligning them in the assembly is not statistically tractable. And also they don't necessarily encode for relevant genes so they are often filtered out. Furthermore, they are arguably the regions of the DNA that is the most prone to change, since you can think of them as the "filler text" of the document; if an important part of the document is unintelligible, then this document wouldn't survive nature's QC process of sexual selection. However, unintelligible parts of the document don't encode important genes, so they evolve very quickly because they have a higher tolerance of typo's and mutations. So in an population pool that people use to assemble and come up with an consensus genome (keep in mind the population pool will have different gene variation), so it may turn out at a particular base pair position in the document, there is so much variation that the genome assembler can't conclusively come up with an consensus sequence or a reasonable distribution of base pair (A, T, C, G).

ende 11y ago

That would be ideal. Unfortunately sequencing is imperfect and there are many points throughout the process where error can be introduced. The easiest source of error is simple contamination. It happens even to the best of bench biologists.

88e282102ae2e5b 11y ago

Not really. If you want tardigrade DNA, you have to break open their cells one way or another. In the pre-print referenced in the article, they basically just froze the tardigrades to -80°C and mechanically smashed them. The problem is that bacteria are pretty fragile. So if you have a tardigrade that has some bacteria sitting on it (or the tardigrade just ate some), whatever process you use to extract the tardigrade DNA is also going to extract the bacterial DNA. Since this is done in solution, the bacterial and tardigrade DNA will be irrevocably mixed. While they do wash and filter the tardigrades extensively and also starve them for several days before extracting their DNA, that's still not enough to completely remove all bacteria.