I’m curious about the whole sequencing process.
Ok, I’m trying to map the whole process. Jee, if you could please pass it onto the sequencing team to see if I’m wrong in any places? I know there are gaps of knowledge that I am missing. Please correct me or fill in the gaps, I think it would be very informative
Please be as exhaustive as you can. Assume that I am going to try doing this, and a step by step basis, would be helpful.
The below are a set of assumptions I have about how we get to the final scoring process. Most of it hasn’t been verified yet.
- RNA sequence winner has been selected
- Goes to the lab, where it get converted to a DNA sequence with U’s turned to T’s.
- You order the oligonucleotide of the DNA sequence. So that’s the opposite of the proposed DNA sequence. eg. G is now a C, and A is now a T.
In eterna’s case, I’m guessing they break up the sequence into two parts, maybe abut 70 bp, then re-assemble at a later point. Hence the second band, when it appears, signalling inaccurate assembly.
- Transcribe into RNA, you do this by putting the seperate oligo DNA strands into a mixture with RNA polymerase and with dNTP (diNucleotideTriPhosphates) which is a free mix of DNA bases.
- Electrophesis, to measure the RNA’s that form and of their lengths
- Purification, only keep the band that we are seeking
- Apply SHAPE with NMIA to the band that is left.
How do you re-assemble the sequences? Ligation?
And in general, I’m not sure how you carry out steps 5, 6 and 7.
Building a DNA molecule
IDT Order Page
picomole is 10^-12 (1 divided by -10 to 12th power)
Only need 3 picomoles for SHAPE to work
umole is 10^6 mole
nmole is 10^9 mole
nanomole is 10^13 mole
micromole is10^16 mole
Avogadro = 6.022
Oh and massive thanks to alan.robot for his contribution to this post!
Here is a brief summary of the process (positive and negative controls are also performed, but not covered in this summary):
- Winning RNA sequences are selected.
- RNA sequence is converted into a DNA sequence by changing U’s to T’s and a 20bp T7-promoter is added to the start of the sequence (the side that starts with GG). This promoter is not transcribed into RNA so it will not affect the folding. http://en.wikipedia.org/wiki/T7_RNA_p…
- Instead of ordering the entire sequence of DNA, which would be very costly and time-consuming, we use Assembly PCR to combine smaller pieces of DNA into the desired full-length strand. We calculate the optimal oligonucleotides to use and order them. http://en.wikipedia.org/wiki/Polymera…
- Once the oligos arrive, we perform Assembly PCR.
- Purify the PCR products.
- Use gel electrophoresis to check for accurate assembly. http://en.wikipedia.org/wiki/Gel_elec…
- Quantify the amount of DNA produced.
- Transcribe the DNA into RNA using a T7 RNA polymerase.
- Purify the transcription products.
- Quantify the amount of RNA produced.
- Perform SHAPE chemical probing using NMIA on RNA.
- Use Reverse Transcription on chemically-modified RNA. Reverse transcriptase stops at bases exposed to NMIA (unpaired bases), producing a band of DNA of a specific length that correlates with the position of an exposed base.http://en.wikipedia.org/wiki/Reverse_…
- While the SHAPE reaction is being performed, create a sequencing ladder of the same RNA to ensure that the correct sequence was produced. http://en.wikipedia.org/wiki/DNA_sequ…
- Use capillary electrophoresis to separate bands created by SHAPE and by sequencing. http://en.wikipedia.org/wiki/Capillar…
- Analyze the SHAPE and sequencing data and upload to EteRNA.
Thank you for that brief summary. Very much appreciated.
I think I can work without controls for now.
If there is enough information, I’m going to try replicate your results.
A few more questions to clarify please. Maybe you could use the current shape, the Bulged Star as the test, and record the results you get from the next upcoming lab designs?
So if you break up the DNA into smaller pieces, how do you break them and how many bases is it?
How do you calculate the optimal oligo’s to use?
How do you join the oligo’s together in Assembly?
How do you purify the PCR products and transcription products?
What do you mean by Quantify, amount of DNA/RNA produced? Is it like 10 DNA strands were produced or like 10% of the solution is of the desired length?
This information with perhaps some additional info on how raw numbers for shape are determined and the new barcode used in cloud lab should be added to omei’s shape wiki note and any info from jnicol’s lab process notes. Then we would have the “synthesis process” detailed out pretty good.
Hyphema, this is the description of the pre-cloud procedure. At a high level – creation of DNA templates, amplification with PCR, transcription, chemical probing, reverse transcription (but not capillary electrophoresis) – things are the same, but most of the details have changed.
Thanks omei. So as Daniel cantu has nicely numbered the steps can you tell me which steps are changed or eliminated? I realize parts are different due to the new cloud lab but thought the early steps were similar. I would like to make a basic stepwise list up to scoring of the lab process but perhaps I am a bit ill-educated to handle this task.
Well, Daniel’s steps 1, 2, 5, 9 and 15 are probably the only ones that haven’t changed in one way or another.
The protocol outline in the wiki is, to the best of my ability, accurate. What needs to be done is to fill in more details. Three of those steps – amplification, transcription and reverse transcription – are standard molecular biology lab procedures. If you want to help document the lab protocol for everyone, perhaps you could fill in some text for those sections, either from your personal knowledge or from Wikipedia. (The PCR is vanilla PCR now, not the Assembly PCR that was being used 3 years ago.)
I’ll continue to fill in details in the parts that are more specific to Eterna, as I continue to understand them better. But It’s always easiest for me to work on something when other people are interested and involved, too. (Hint. )