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| jrayhawk | http://mirror.linux.org.au/pub/linux.conf.au/2012/Keynote_Bruce_Perens.ogv | 08:14 |
|---|---|---|
| kanzure | what is it about | 08:20 |
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| archels | http://vimeo.com/8569187 | 09:13 |
| jrayhawk | Some combination of open hardware or ideological warfare | 09:15 |
| jrayhawk | s/or/and | 09:15 |
| jrayhawk | and lots of other stuff in there is interesting | 09:20 |
| jrayhawk | http://mirror.linux.org.au/pub/linux.conf.au/2012/ that is | 09:20 |
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| kanzure | http://joostn.github.com/OpenJsCad/ | 10:46 |
| kanzure | although it's just boolean ops on meshes still (csg.js) | 10:46 |
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| kanzure | gah | 10:50 |
| kanzure | some guy is trying to argue that OpenJsCad is boolean operations on NURBS | 10:50 |
| kanzure | so not only do i have to continue to explain that blender is not the same sort of engine as solidworks or catia, | 10:51 |
| kanzure | but the developers don't even know the difference | 10:51 |
| kanzure | this probably is insufficient | 10:55 |
| kanzure | http://diyhpl.us/wiki/cadfaq | 10:55 |
| kanzure | i suppose most people don't care if their models are 100s of thousands of tessellated triangles, as long as it prints on the printer.. (yawn) | 10:56 |
| kanzure | csg.js is nice if you never, ever share the output of the file, but only the source file; also, it's nice if you never, ever actually render large models with it | 10:57 |
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| kanzure | hah | 11:08 |
| kanzure | http://pubs.rsc.org/en/Content/ArticleLanding/2011/LC/c0lc00577k | 11:08 |
| kanzure | so this seems to use an optical method to guide beads into different areas for dna synthesis | 11:08 |
| kanzure | i'm not sure how it prevents the different reservoires from mixing into the main channel | 11:09 |
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| delinquentme | so is it a difficult thing to come up with novel math applications in bio? | 11:18 |
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| kanzure | delinquentme: yes, it's very difficult to know whether or not someone has previously thought about a certain math concept | 11:19 |
| delinquentme | kanzure, SO i guess its correct to say that in order to apply mathematical models effectively to these problems you've got to have a solid basis in math processes | 11:21 |
| delinquentme | IE knows lots about the applications | 11:21 |
| kanzure | what? | 11:21 |
| delinquentme | you've got to know lots of math | 11:22 |
| delinquentme | in order to pickout which model / technique | 11:22 |
| delinquentme | would best help a problem | 11:22 |
| kanzure | sure. or you can just make up the math on your own (this has the downside of making it hard to search for what other people call the problem, or how they approached similar problems) | 11:23 |
| kanzure | wow wow, what | 11:23 |
| kanzure | why is elsevier displaying ads on sciencedirect ow | 11:23 |
| kanzure | *now | 11:23 |
| kanzure | scroll down to the bottom on http://www.sciencedirect.com/science/article/pii/S0003269710004045 | 11:23 |
| delinquentme | ya thats quite the hefty popup | 11:24 |
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| kanzure | hrm. it occurs to me that it should be possible to spam google scholar's index, | 11:27 |
| kanzure | for every paper that sciencedirect hosts, generate a pdf with the same citation information | 11:27 |
| kanzure | then allow google scholar to crawl these pdfs | 11:28 |
| kanzure | instead of content, the pdfs will be something else | 11:28 |
| kanzure | i'm fairly confident that nobody in their right mind goes to sciencedirect except from google scholar | 11:28 |
| delinquentme | ok here we go: | 11:30 |
| delinquentme | a program to check whether particular beneficial proteins are expressed in microarray data | 11:31 |
| kanzure | can i convince you to just work on a dna synthesis instrument instead :P | 11:32 |
| delinquentme | kanzure, what are you doing with it :D | 11:37 |
| delinquentme | I agree that DNA synthesis will be huge soon | 11:38 |
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| kanzure | delinquentme: well, my ideal scenario is a polymerase that i can control | 11:43 |
| kanzure | deliquentme: but i'd settle with a microfluidic or desktop-sized device that would build oligos of any length | 11:43 |
| kanzure | possibly from a library, or possibly from de novo synthesis | 11:43 |
| delinquentme | well how do you get the polymerase to work | 11:44 |
| kanzure | it would probably require on-board sequencing for verification | 11:44 |
| kanzure | nobody knows that yet, so a more traditional dna synthesis approach is needed | 11:44 |
| delinquentme | kanzure, sounds like you should just build a system which consistently sequences the right bases | 11:44 |
| kanzure | what? i'm talking about synthesis | 11:45 |
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| delinquentme | kanzure, yeah I mean skip the expensive machine | 11:46 |
| Lucas_ | good afternnon | 11:46 |
| delinquentme | so how to isolate the polymerase? | 11:46 |
| kanzure | isolating polymerase isn't an issue | 11:46 |
| delinquentme | but doesnt the polymerase use a single strand to create sequences? | 11:46 |
| Lucas_ | ohh, biology | 11:46 |
| delinquentme | ergo you'd already have a particular sequence synthesized in order for it to work? | 11:46 |
| Lucas_ | what's the issue, I am curious | 11:47 |
| delinquentme | :P | 11:47 |
| kanzure | delinquentme: are you talking about traditional dna synthesis, or yesterday's crazy polymerase hacking work? | 11:47 |
| delinquentme | Lucas_, kanz is interested in DNA synthesis using really fast tools | 11:47 |
| delinquentme | kanzure, im just asking questions as to how the polymerase would synthesize DNA | 11:47 |
| kanzure | once you synthesize a 6nt strand, you still need to combine it with the next strand, thus polymerase.. pretty standard | 11:47 |
| delinquentme | ( because im 95% clueless ) | 11:48 |
| kanzure | ok, are you asking how pcr works? | 11:48 |
| Mariu | you reminded me of 'The Architect' from 'The Matrix', when you said "ergo" xD | 11:48 |
| delinquentme | nah im fairly comfortable w themocycling | 11:48 |
| kanzure | thermocycling is just a process... | 11:48 |
| delinquentme | Mariu, :D | 11:48 |
| Mariu | =p | 11:48 |
| kanzure | pcr is polymerase chain reaction. it uses polymerase. | 11:48 |
| delinquentme | kanzure, true but you're talking "from scratch" synthesis | 11:49 |
| kanzure | so anyway, let's say you do de novo oligo synthesis of 6 nt, you still need to sequence and confirm that it's valid, then you do ligation+pcr | 11:49 |
| delinquentme | PCR is simply amplification of an existing sequence | 11:49 |
| delinquentme | 6 nt | 11:49 |
| delinquentme | nucleotides | 11:49 |
| delinquentme | kk | 11:49 |
| kanzure | bp | 11:49 |
| kanzure | usually you do single-strand synthesis.. so bp isn't accurate, but people know what you're talking about | 11:50 |
| delinquentme | kanzure, you dont need ligation | 11:50 |
| delinquentme | you're doing denovo synthesis ... there are no cells to rupture | 11:50 |
| delinquentme | right? | 11:50 |
| kanzure | ligation is the joining of two dna strands together using something like dna ligase | 11:51 |
| delinquentme | you're talking like a machine which legos this shit together and viola you've got 6 nucleotides | 11:51 |
| delinquentme | LIGATION not lysing ok | 11:51 |
| kanzure | um, yeah- de ovo oligo synthesis is very standard | 11:51 |
| delinquentme | ok | 11:51 |
| delinquentme | im with you .. continue | 11:51 |
| kanzure | you can run the phosphoramidite method for as long as you want, making 1000s of bp of dna | 11:51 |
| kanzure | but there is a natural error rate due to problems | 11:52 |
| kanzure | sometimes that's 1 in 250 or worse | 11:52 |
| kanzure | polymerase does 1 error in 10,000 or better | 11:52 |
| kanzure | so if yuo want to be sure that your output is correct in the process of synthesis, you sequence it along the way | 11:53 |
| kanzure | http://diyhpl.us/~bryan/papers2/microfluidics/synthesis/microfluidics_Quake_DNA_synthesizer-20mers-PFPE.png | 11:53 |
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| kanzure | then you do onboard sanger sequencing of anything you synthesize http://diyhpl.us/~bryan/papers2/microfluidics_Sanger_sequencer.png | 11:54 |
| delinquentme | so then your polymerase hangs out in the reaction chambers | 11:56 |
| kanzure | sure | 11:57 |
| delinquentme | and you pump in the nucleotides through the channels | 11:57 |
| delinquentme | Is that sanger sequencing microfluidics technique not patented? | 11:57 |
| kanzure | i'm sure someone has patented sanger sequencing | 11:57 |
| kanzure | and i'm sure someone has patented a form of sanger sequencing on microfluidic chips | 11:58 |
| delinquentme | so how do current companies run their synthesis process? | 12:03 |
| delinquentme | like mrgene | 12:03 |
| kanzure | i think they outsource to dit :P | 12:05 |
| kanzure | *IDT | 12:05 |
| kanzure | but no, there's "dna synthesis machines" that use phosphoramidite synthesis or some other bead-based method | 12:06 |
| delinquentme | so then if you've got polymerase and you've got a source for the fabrication of the chips .. what else do you need? | 12:06 |
| kanzure | no, i'd have to build the chips | 12:07 |
| kanzure | and build the oligo library, and some way to let people restock their oligo library.. hrm | 12:07 |
| delinquentme | you' build the chips? | 12:09 |
| delinquentme | that sounds nuts IMO | 12:09 |
| delinquentme | why? | 12:09 |
| delinquentme | chemical etching? | 12:10 |
| kanzure | first of all, the chips don't exist | 12:10 |
| strages_work | or if you have a laser.... | 12:10 |
| kanzure | it'd probably be pdms photolithography stuff | 12:10 |
| kanzure | or laser cut | 12:10 |
| kanzure | not a big deal | 12:11 |
| delinquentme | strages_work, true but I dont think kanz has this laying around? | 12:11 |
| kanzure | um, i do have a laser cutter | 12:11 |
| strages_work | a local hackerspace might.... | 12:11 |
| kanzure | i'm the one who bought the laser cutter for the austin hackerspace (at least, the first laser cutter) | 12:11 |
| kanzure | (not the one they are currently using at their new location) | 12:12 |
| delinquentme | IMO it shoulds like shit you should just outsource | 12:12 |
| delinquentme | and its not like you need to customize these | 12:12 |
| kanzure | outsource the design of this device? | 12:12 |
| delinquentme | you just need a number of them bc what you're doing it __NOT__ designing novel experiments but instead synthesizing | 12:13 |
| kanzure | you mean the fabrication? no, i think for prototyping i should do fabrication | 12:13 |
| delinquentme | kanzure, dont you have the design? | 12:13 |
| kanzure | nope, i thought this is what we were talking about | 12:13 |
| delinquentme | ah ok so you're at the design point here | 12:15 |
| delinquentme | so you need to come up with some mechanism which would allow the polymerase to build the DNA | 12:15 |
| delinquentme | but im still missing the key aspect | 12:15 |
| delinquentme | polymerase is synthesis by sequenceing | 12:16 |
| delinquentme | is it not? | 12:16 |
| delinquentme | how does that help you with de novo snyth? | 12:16 |
| kanzure | what? | 12:17 |
| kanzure | are you confusing two different goals of polymerase protein engineering vs. dna synthesis? | 12:17 |
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| delinquentme | kanzure, what you're trying to do is dna snythesis right? | 12:19 |
| kanzure | de novo synth can happen without polymerase, but you still need to copy the resulting output so that it is usable | 12:19 |
| delinquentme | you want insilico >> ACCACATAGAGTA>> machineX >> chemical ACCACATAGAGTA | 12:20 |
| kanzure | (pcr) | 12:20 |
| kanzure | yes, there's already many well known mechanism of de novo dna synthesis and strategies for dna assembly (without de novo synthesis) | 12:21 |
| delinquentme | if what you're trying to do is take in sequences and then have a machine assemble a chemical bonded corresponding molecule based off of that sequence we're on the same page | 12:21 |
| kanzure | the trick is picking which one, designing a microfluidic device to actually do it properly, etc. | 12:21 |
| delinquentme | on cool | 12:21 |
| delinquentme | but then I dont get how polymerase helps you? | 12:21 |
| delinquentme | polymerase reads an existing chunk of DNA right? | 12:21 |
| kanzure | are you asking "how does yesterday's chat about polymerase engineering help you?" | 12:22 |
| delinquentme | ohhhhh | 12:22 |
| kanzure | or "why do you need pcr after dna synthesis?" | 12:22 |
| delinquentme | the polymerase is not part of this synthesis operation? | 12:22 |
| delinquentme | im trying to figure out the mechanism you're wanting to use for todays chat about synthesis | 12:22 |
| kanzure | well, you can use polymerase if you're doing bead-based synthesis (like your strand is attached to the bead); this is also the same bead strategy in phosphoramidite synthesis heh | 12:23 |
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| delinquentme | can i convince you to just work on a dna synthesis instrument instead :P << kanzure | 12:53 |
| delinquentme | is this instrument using polymerase | 12:54 |
| kanzure | depends on the design that we choose | 12:55 |
| delinquentme | ah! ok | 12:56 |
| delinquentme | now i comprendo | 12:56 |
| delinquentme | arent microfluidic chips lined with some kind of fluid? | 12:56 |
| delinquentme | like the fluids runing through the chips .. do they actually come in contact with the plastic | 12:57 |
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| kanzure | delinquentme: yes. the plastic has channels. the fluid runs through the channels. | 12:58 |
| delinquentme | kanzure, yeah i thought I read somewhere that those fluids are suspended w something else | 12:59 |
| kanzure | they can be | 12:59 |
| kanzure | like water-in-oil | 12:59 |
| delinquentme | yeah! | 12:59 |
| delinquentme | ok not nuts | 12:59 |
| kanzure | which makes it "digital" microfluidics (droplets) | 12:59 |
| delinquentme | Ohhh ok ok | 13:01 |
| delinquentme | yeah i've seen that before | 13:01 |
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| delinquentme | howdy bio_boris =] | 13:03 |
| kanzure | hi bio_boris | 13:03 |
| delinquentme | kanzure, i just posted in r/bioinformatics about a bunch of channels | 13:03 |
| delinquentme | boris found it :D and checked us out | 13:03 |
| bio_boris | hello | 13:04 |
| delinquentme | bio_boris, are you a programmer? | 13:04 |
| bio_boris | yea guess so | 13:04 |
| bio_boris | perl | 13:04 |
| bio_boris | im a masters student studying bioinformatics | 13:05 |
| kanzure | have a thesis? | 13:05 |
| bio_boris | nope | 13:06 |
| delinquentme | oh bad ass | 13:06 |
| bio_boris | elected the non thesis option lol | 13:06 |
| delinquentme | theres a non thesis option?? | 13:06 |
| bio_boris | Yea its a 'professional masters' | 13:06 |
| bio_boris | rather | 13:06 |
| bio_boris | from a 'Professional School' | 13:06 |
| delinquentme | oh thats cool | 13:07 |
| delinquentme | germany? | 13:07 |
| bio_boris | http://www.lis.illinois.edu/academics/programs/ms-bioinformatics | 13:07 |
| delinquentme | is perl their chosen language? | 13:07 |
| bio_boris | its what ive used the most so far | 13:07 |
| delinquentme | bio_boris, what tetbook are you using? hows the homework? | 13:08 |
| bio_boris | i have a few textbooks, class just started last week so not too much homework yet | 13:08 |
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| bio_boris | im actually working on research now, tryin to remove UTRs from a FASTA file from a GFF file | 13:09 |
| bio_boris | (using a gff3 file) | 13:09 |
| delinquentme | bio_boris, ooooo!!! | 13:10 |
| delinquentme | talk to me about this i've been working w GFF3 files | 13:10 |
| delinquentme | and wouldn't that be just checking the 9th column for a Keyword? | 13:10 |
| bio_boris | I have a list like this now | 13:11 |
| delinquentme | bio_boris, are you working w a lab? | 13:11 |
| bio_boris | MRNA start stop seqid | 3prime utr start stop seqid | 3prime utr start stop seqid | 5prime utr start stop seqid | 13:11 |
| bio_boris | so i need to do a little tiny bit of math and grab out some substrings from the fasta files | 13:11 |
| bio_boris | of MRNA | 13:12 |
| delinquentme | so wait are you calculating which region is the UTR? | 13:12 |
| bio_boris | the gff says so , like | 13:12 |
| bio_boris | MRNA 7000 to 7030 | 5 prime UTR 7000 to 7005 | 5 prime UTR 7010 to 7015 | 13:13 |
| bio_boris | so i want 7006-7009+7016-7030 | 13:14 |
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| bio_boris | i want the MRNA sequence from positions 7006 to 7009 and 7016 to 7030 | 13:15 |
| bio_boris | about to write something now to try to do that | 13:15 |
| delinquentme | hmmm thats a legit gff3 format? | 13:15 |
| delinquentme | where did you pick those up? | 13:15 |
| bio_boris | i took the GFF3 data and converted it | 13:15 |
| bio_boris | to something easier to work with | 13:15 |
| delinquentme | Ahhh ok | 13:15 |
| bio_boris | cuz the GFF file was too big | 13:15 |
| bio_boris | i only needed the start, stop, feature and description | 13:15 |
| delinquentme | where did you get the original GFF3 from? | 13:15 |
| bio_boris | utah.edu | 13:16 |
| delinquentme | do they have a repo of gff3 files? | 13:16 |
| bio_boris | im not sure, its from a particular lab | 13:16 |
| bio_boris | working on a particular project | 13:16 |
| delinquentme | ive been bug testing biopythons GFF parser | 13:16 |
| bio_boris | Gff3 == Gff ? | 13:17 |
| bio_boris | guess not | 13:18 |
| delinquentme | so they're slightly different | 13:20 |
| delinquentme | whats really weird is one place maintains the standards for gff2 | 13:20 |
| delinquentme | and another gf3 | 13:20 |
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| ybit | jrayhawk: oi | 15:12 |
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| gene_hacker | you there kanzure? | 17:02 |
| kanzure | gene_hacker: hi | 17:03 |
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| delinquentme | gene_hacker, kanzure no sleep | 17:16 |
| kanzure | no who? | 17:20 |
| kanzure | he's pming me :( | 17:20 |
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| delinquentme | kanzure, you looking for a good time? | 17:27 |
| delinquentme | gene_hacker, share the luv | 17:27 |
| kanzure | hi aristarchus | 17:28 |
| gene_hacker | you'd have a huge tangle of rope attached to the ground, blowing in the wind | 17:28 |
| aristarchus | kanzure: hello | 17:28 |
| kanzure | gene_hacker: nah, not an issue | 17:28 |
| kanzure | that's how it currently works | 17:29 |
| gene_hacker | when your strand get's up to size HUGE! wouldn't it get washed out too? | 17:29 |
| kanzure | no, it's attached to the bead | 17:29 |
| kanzure | (or to the wall) | 17:29 |
| gene_hacker | so in some sort of gel? | 17:29 |
| kanzure | nope.. one sec | 17:30 |
| gene_hacker | so are you combining oligo in parallel? or in one large chamber? | 17:30 |
| kanzure | well, i'm not sure, i think parallel would be smart, but probably hard to manage | 17:30 |
| kanzure | so let's say you combine A+B | 17:31 |
| kanzure | you need to verify that A+B worked, and then store the result until you know the results | 17:31 |
| kanzure | like if you do on chip sequencing | 17:31 |
| kanzure | now, you might not want to sequence after every operation so maybe it's A+B+C+D+E that you want to test | 17:31 |
| kanzure | and then you combine (A+B+C+D+E) with (F+G) | 17:31 |
| gene_hacker | so one central bead where you apply oligo one at a time? | 17:32 |
| kanzure | so you'd have maybe 100 different addressable "banks" to temporarily store stuff in | 17:32 |
| kanzure | gene_hacker: where you apply the +5 bp, yes.. | 17:32 |
| kanzure | multiple chambers/reactions is possible but it complicates the circuit design | 17:32 |
| kanzure | gene_hacker: http://diyhpl.us/~bryan/papers2/microfluidics/synthesis/Synthesis%20-%20Integrated%20two-step%20gene%20synthesis%20in%20a%20microfluidic%20device%20(1k%20bp,%201%20error%20per%20250%20bp).pdf | 17:33 |
| kanzure | this is an ok example i think | 17:33 |
| gene_hacker | are you put the dna together 5 bp at a time? | 17:34 |
| kanzure | yes | 17:35 |
| kanzure | but you can imagine storing the "result" of 5+5 somewhere, and later using it | 17:36 |
| kanzure | or even "1000+5", and using that later | 17:36 |
| gene_hacker | in the paper you mention, they put together 40 mer oligos with 20 mer overlap | 17:36 |
| kanzure | yeh, this paper is slightly different | 17:36 |
| gene_hacker | how'd they get those oligos? | 17:36 |
| kanzure | i haven't read this one in a while; it sorta depends | 17:37 |
| kanzure | they might have synthesized those on a dna microarray with photochemistry | 17:37 |
| gene_hacker | so it is difficult to parallelize your method? | 17:37 |
| gene_hacker | so how long would one step take? | 17:37 |
| kanzure | that depends on channel geometry | 17:38 |
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| gene_hacker | what's the most reasonable estimate for how long adding one oligo would take | 17:38 |
| kanzure | i'm looking this up, i don't want to bs oyu | 17:38 |
| gene_hacker | 1 second, ten seconds, 30 seconds? | 17:38 |
| kanzure | *you | 17:38 |
| kanzure | i'd say 10-30 seconds is a good target to reach | 17:39 |
| kanzure | but currently there's no commercial synthesizer that does anything near 1 bp/sec | 17:39 |
| delinquentme | i learned about sample std dev and population std dev | 17:39 |
| gene_hacker | about an hour for a kilobase | 17:40 |
| kanzure | hmm | 17:40 |
| gene_hacker | well I need to go | 17:40 |
| gene_hacker | that's not bad | 17:40 |
| kanzure | i'm pretty sure it's worse than that | 17:41 |
| kanzure | but | 17:41 |
| kanzure | small reaction sample sizes might allow you to increase reaction rates | 17:41 |
| kanzure | like with pcr (nanoliter droplet + laser => orders-of-magnitude-faster pcr) | 17:41 |
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| gene_hacker | the other problem is the rate of consumption of your oligos | 17:41 |
| kanzure | this is why they need to be pcr tubes (or something else), | 17:42 |
| kanzure | so that you can replace your stock | 17:42 |
| gene_hacker | your stock could be expensie | 17:42 |
| kanzure | yep probably | 17:42 |
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| kanzure | hi shipwreck__ | 17:42 |
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| aristarchus | what are some good diy h+ sites? | 18:03 |
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| kanzure | aristarchus: depends on what you want.. | 18:12 |
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| aristarchus | this might be a chicken/egg problem | 18:16 |
| kanzure | well, then i have no trouble saying this is the best diyh+ site on the web :) | 18:17 |
| kanzure | with the best people. | 18:18 |
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| aristarchus | haha | 18:21 |
| aristarchus | indeed | 18:21 |
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| aristarchus | right now i'm looking for hi-tech home improvement type stuff | 18:26 |
| aristarchus | like controlling your thermostat from your phone | 18:26 |
| minu | biohacking <- I like this term | 18:26 |
| sylph_mako | Every house needs more of that. | 18:29 |
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| kanzure | hi yashgaroth | 18:34 |
| yashgaroth | whatup kanzure | 18:35 |
| kanzure | software errors | 18:35 |
| kanzure | this assembler i'm working with, | 18:35 |
| kanzure | doesn't let code in for loops, access the global variables | 18:35 |
| kanzure | i.e. all variables are reset.. hrm | 18:35 |
| yashgaroth | I know just enough programming to know what that problem entails | 18:37 |
| eudoxia | don't some languages have special syntax for global variables? | 18:37 |
| kanzure | not this one | 18:37 |
| kanzure | eudoxia: http://otakunozoku.com/RGBDSdocs/ | 18:37 |
| kanzure | oops, http://otakunozoku.com/RGBDSdocs/asm.htm | 18:38 |
| eudoxia | oh, for that pokemon project= | 18:38 |
| eudoxia | ? | 18:38 |
| kanzure | heh | 18:39 |
| kanzure | don't judge me | 18:39 |
| eudoxia | you got my undying respect with that copy of Nanosystems | 18:40 |
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| aristarchus | oooh rom hacking? | 18:40 |
| kanzure | aristarchus: i'm writing source code to pokemon red | 18:40 |
| kanzure | https://bitbucket.org/kanzure/pokered/changesets | 18:41 |
| aristarchus | cool | 18:43 |
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| gene_hacker | whoa you have nanosystems? | 19:57 |
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| kanzure | gene_hacker: sure | 19:58 |
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| eudoxia | I can't access /stuff_to_deal_with/nanosystems.tar.gz though | 19:58 |
| gene_hacker | where? | 19:59 |
| kanzure | why not | 19:59 |
| kanzure | http://gnusha.org/stuff_to_deal_with/nanosystems.tar.gz | 19:59 |
| kanzure | try now.. | 20:00 |
| gene_hacker | forbiden | 20:00 |
| eudoxia | 403 forbidden | 20:00 |
| kanzure | try one more time. | 20:01 |
| kanzure | but this time wish reallllly hard | 20:01 |
| eudoxia | it works now | 20:01 |
| eudoxia | hahahahahah | 20:01 |
| gene_hacker | so is it possible to buy short 5 bp oligo nucleotides like you mentioned? | 20:02 |
| kanzure | yes you can buy sequences of any length you want | 20:02 |
| kanzure | http://mrgene.com/ | 20:02 |
| kanzure | huh invitrogen bought mrgene? | 20:03 |
| kanzure | $0.35/bp .. why is it increasing in price | 20:03 |
| kanzure | that's all wrong | 20:03 |
| gene_hacker | but is it possible to make very pure 5 pb segments? | 20:05 |
| gene_hacker | http://www.lightlabsusa.com/Rota-Rack-Duo-Tube-Rack.html | 20:06 |
| kanzure | yes they will give you a high quality sample.. i mean, that's their job :p | 20:06 |
| gene_hacker | now this is a tube rack | 20:06 |
| gene_hacker | how much would 1024 different samples cost though? | 20:06 |
| kanzure | a lot. you would not order 1024 samples from them | 20:06 |
| kanzure | you'd probably setup a custom business outfit to make oligo librarise- or get better pricing from somewhere else | 20:07 |
| gene_hacker | from the dimensions on the tube rack, 32x32 eppie tubes would take up 2'x2' | 20:07 |
| gene_hacker | that's pretty big | 20:07 |
| kanzure | once you have the initial library it's really easy to just copy as much as you need | 20:07 |
| kanzure | 2 feet? bah | 20:07 |
| kanzure | not bad | 20:07 |
| gene_hacker | that's pretty big | 20:07 |
| kanzure | you could keep it under your bed :P | 20:08 |
| gene_hacker | Now how would you dispense nanoliters of droplets from the epie tube? | 20:08 |
| kanzure | not sure, haven't thought about that yet | 20:08 |
| gene_hacker | 1 valve per tube? | 20:08 |
| kanzure | probably | 20:08 |
| gene_hacker | so you have 1024 valves | 20:08 |
| kanzure | minimum | 20:08 |
| kanzure | there also has to be either (1) dedicated channels for each tube or (2) a way to wash the whole thing after each withdrawal | 20:09 |
| kanzure | s/wash/flush | 20:09 |
| gene_hacker | that sounds complex | 20:09 |
| kanzure | yes.. so #1 is preferable ;) | 20:09 |
| gene_hacker | how would one replace a valve when it fails | 20:10 |
| gene_hacker | still you're going to need moving parts | 20:10 |
| kanzure | i haven't come up with an optimal solution yet, i'm open to ideas | 20:10 |
| gene_hacker | plus your nanoliter droplets need to travel over 2 feet to get to where they need to go | 20:10 |
| yashgaroth | oh ffs you can at least use multiwell plates | 20:11 |
| kanzure | not if you want a person to replace each well | 20:11 |
| kanzure | replenish each well | 20:11 |
| gene_hacker | I don't think you can do that with pipes over that distance, at least not without using a bunch of expensive fluid | 20:11 |
| yashgaroth | replenishment is no harder with a well if all you're doing is adding more | 20:12 |
| kanzure | you can't expose the liquid to atmosphere | 20:12 |
| kanzure | ever.. | 20:12 |
| gene_hacker | not even helium? | 20:12 |
| yashgaroth | what why not? | 20:12 |
| kanzure | i'd prefer not to work with helium or gases | 20:12 |
| kanzure | yashgaroth: contamination | 20:12 |
| yashgaroth | run the whole thing in a TC hood, those things are great | 20:12 |
| kanzure | it's much easier if you just don't use atmosphere | 20:12 |
| yashgaroth | I literally don't even need to use gloves, never had contamination in a thousand flasks | 20:13 |
| kanzure | anyway, i don't really trust users to refill a 32x32 microarray with the right oligos | 20:13 |
| gene_hacker | Could you breed oligos on the spot? | 20:14 |
| yashgaroth | oh, me either, but that's not a case for using ep tubes | 20:14 |
| gene_hacker | from common compounds | 20:14 |
| kanzure | gene_hacker: yes, this is called "oligonucleotide synthesis" | 20:14 |
| kanzure | it's a five or six step process per bp | 20:15 |
| gene_hacker | then what benefit does the 1024 library have over conventional oligonucleotide synthesis? | 20:15 |
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| kanzure | you don't have to synthesize any of the 1024 :P | 20:15 |
| gene_hacker | then how do you get more? | 20:15 |
| kanzure | 1) mail order | 20:16 |
| kanzure | 2) pcr | 20:16 |
| kanzure | either option. | 20:16 |
| gene_hacker | so you're just using someone else's error prone oligonucleotide synthesis? | 20:16 |
| kanzure | no, you'd get someone who does verification of their sequences | 20:16 |
| kanzure | i.e. someone who synthesizes then sequences to verify | 20:17 |
| gene_hacker | isn't oligonucleotide synthesis inherently error prone? | 20:17 |
| yashgaroth | you can't really sequence an oligo that short, and even then it's always the 0.1% that's gonna be wrong | 20:17 |
| kanzure | but yes, oligo synthesis does have a high error rate | 20:17 |
| kanzure | that's why you want to get that step out of the way | 20:17 |
| kanzure | and just use a library of known good strands | 20:17 |
| gene_hacker | a library of 1024 unique parts that each have to be inspected before use? | 20:18 |
| gene_hacker | that sounds expensive | 20:18 |
| yashgaroth | couldn't you just mix a load of 6-mers together and ligate into strands? like 3 & 3 overlapping | 20:19 |
| kanzure | yes, but you just inspect it once, | 20:19 |
| kanzure | so let's say that you don't want to pcr your library contents when they get low, | 20:19 |
| kanzure | you'd just ask me to send over some more, and i'd do the pcr from my stock :p | 20:19 |
| kanzure | yashgaroth: yes but you want to keep them as 6mers really | 20:19 |
| yashgaroth | well yes | 20:20 |
| kanzure | so you need to keep them separated | 20:20 |
| yashgaroth | I | 20:20 |
| yashgaroth | 'm not saying mix everything at once | 20:20 |
| kanzure | now, if you have a mixed library, | 20:20 |
| kanzure | you'd need some way to address individual items in the library (like with dna) | 20:20 |
| kanzure | so you'd synthesize your "caller".. etc. | 20:20 |
| kanzure | anyway.. i think a separated library is a better idea | 20:20 |
| yashgaroth | but if you make a ~30bp strand where none of the wrong 6mers would overlap each other | 20:20 |
| kanzure | i could be convinced of doing on-chip oligo synthesis, and not having a library, but this just seems really error prone.. you'd definitely need on-chip sequencing, | 20:21 |
| kanzure | and you'd have to sequence every 20bp chunk you make | 20:21 |
| yashgaroth | nah, if all your 6mers are good and don't overlap the wrong way they should be fine | 20:21 |
| kanzure | presumably you'd select a good 6mer based on the current bases near your extending end | 20:22 |
| yashgaroth | you can extend from both ends | 20:22 |
| gene_hacker | well I guess you're using only 200 nanoliters per kb, it might be doable | 20:22 |
| kanzure | yashgaroth: not if one end is attached to a bead ;) | 20:22 |
| yashgaroth | man screw beads | 20:22 |
| gene_hacker | now how much DNA would one get from a process like this? | 20:22 |
| kanzure | it doesn't matter, | 20:22 |
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| kanzure | you would use pcr to copy the dna | 20:22 |
| gene_hacker | it does | 20:23 |
| kanzure | so let's say you end up with 1 picogram | 20:23 |
| kanzure | you can pcr that up to a few thousand nanograms or whatever you need | 20:23 |
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| gene_hacker | when you want to copy a big sequence of DNA from small amounts you have to go through a whole bunch of special steps | 20:23 |
| yashgaroth | a nanogram is enough for transformation if you know what you're doing btw | 20:23 |
| gene_hacker | I read that the PCR for that is the most difficult step | 20:24 |
| kanzure | pcr is very well known at this point.. i wouldn't worry about it | 20:24 |
| yashgaroth | define 'difficult' | 20:24 |
| kanzure | yashgaroth: http://diyhpl.us/~bryan/papers2/microfluidics/PCR%20-%20Nanodroplet%20real-time%20PCR%20system%20with%20laser%20assisted%20heating.pdf | 20:24 |
| kanzure | 40 cycles in 370 seconds = win | 20:25 |
| yashgaroth | plus, lasers | 20:25 |
| kanzure | and not the "have a big giant co2 tube" kind | 20:25 |
| yashgaroth | pcr really isn't the hard part in this sort of thing anyway | 20:26 |
| kanzure | getting 1 bp error per 100-200 is the hard part | 20:27 |
| kanzure | the problem with on-chip sequencing is that it's a completely different process to debug | 20:27 |
| gene_hacker | well then what's the problem craig venter is having with chromosome construction? | 20:27 |
| kanzure | so not only do you have synthesis (or oligo library stuff), but also sequencing.. yikes | 20:27 |
| yashgaroth | it cost millions upon millions for venter to pull that stunt | 20:28 |
| gene_hacker | are you sequencing the strands as they are made? | 20:30 |
| kanzure | well, if you have on-chip synthesis then yes, you should sequence as soon as you synthesize anything more than.. 10 bp | 20:30 |
| yashgaroth | are you sequencing with PCR? because you'll need a lot more DNA than we'd be working with to get a good signal | 20:31 |
| gene_hacker | is it possible to build an on chip synthesizer? | 20:32 |
| yashgaroth | sure, with photolithography | 20:34 |
| gene_hacker | oops meant sequencer | 20:34 |
| gene_hacker | and would it be possible to move the DNA strand being synthesized around the chip on say a bead or something? | 20:35 |
| yashgaroth | that would be extremely hard to control | 20:35 |
| kanzure | gene_hacker: yes, people have done on-chip synthesis with various methods, though i haven't seen on-chip photo-based synthesis | 20:36 |
| kanzure | the sequencing method would be a very simple sanger sequencing approach, i think | 20:36 |
| gene_hacker | what about picoarray? | 20:36 |
| gene_hacker | that's onchip photo-based synthesis | 20:36 |
| kanzure | yes, it would be possible to move synthesized dna around, like in droplet microfluidics | 20:36 |
| yashgaroth | sanger sequencing won't work on reads that short | 20:36 |
| kanzure | gene_hacker: picoarray? this? http://diyhpl.us/~bryan/papers2/microfluidics/synthesis/Synthesis%20-%20Microfluidic%20PicoArray%20synthesis%20of%20oligodeoxynucleotides%20and%20simultaneous%20assembling%20of%20multiple%20DNA%20sequences%20(10%20kb).pdf | 20:37 |
| gene_hacker | yeah | 20:37 |
| gene_hacker | the photogenerated acid one | 20:37 |
| kanzure | been a while since i've read this one.. | 20:37 |
| gene_hacker | doesn't use any exotic photo-capped oligos | 20:37 |
| gene_hacker | now if you have to wait 30 seconds for PCR to go through, I guess you could move the whole PCR chip to the required eppie tube while PCR is taking place | 20:40 |
| gene_hacker | and use a 1 nanoliter sucker | 20:41 |
| gene_hacker | instead 1024 valves | 20:41 |
| yashgaroth | how the shit are you guys sequencing a 10bp fragment | 20:42 |
| kanzure | i don't know, i haven't come up with that yet | 20:42 |
| gene_hacker | Well you could use some probes to read the ends? | 20:43 |
| kanzure | what? | 20:43 |
| yashgaroth | even primers for sequencing are longer than 10bp | 20:43 |
| yashgaroth | and as a rule of thumb the first 50 bases of sanger sequencing are a write-off | 20:43 |
| gene_hacker | so kanzure are you sure that PCR has a lower error rate than oligosynthesis? | 20:44 |
| yashgaroth | pcr amplification does, yes | 20:44 |
| kanzure | oh man, yeah | 20:44 |
| kanzure | just completely blan out "PCR" as a problem right now | 20:44 |
| kanzure | *blank out | 20:44 |
| kanzure | dna comes in -> more dna comes out | 20:44 |
| kanzure | (plus other crap) | 20:45 |
| gene_hacker | but when you're doing 200 or more amplification steps and your errors accumulate, is the error rate still lower? | 20:45 |
| kanzure | the errors happen in the synthesis stage | 20:45 |
| yashgaroth | that's why you sequence the final product, isolated from a single transformed bacterium | 20:45 |
| kanzure | huh? let's not bring in cell cultures | 20:46 |
| gene_hacker | why is it put into a bacteria? to amplify it I presume? | 20:46 |
| kanzure | gene_hacker: it's just one of the things you might want to do with the dna | 20:46 |
| yashgaroth | yes, and also bacterial amplification is a million times more accurate than PCR | 20:46 |
| yashgaroth | due to a functioning proofreading complex | 20:46 |
| gene_hacker | but it's slow, correct? | 20:46 |
| kanzure | 12 hour incubation period, is my guess | 20:47 |
| yashgaroth | within a day you have unlimited amplification | 20:47 |
| yashgaroth | but yes, it takes a day | 20:47 |
| gene_hacker | and isolation? | 20:47 |
| yashgaroth | trivial | 20:47 |
| gene_hacker | though does it involve manual labor? | 20:47 |
| kanzure | "trivial".. on a chip? you're just making this more work for me :p | 20:48 |
| gene_hacker | like picking glowing colonies? | 20:48 |
| yashgaroth | liter-scale bac culture is trivial | 20:48 |
| yashgaroth | haha if picking colonies is manual labor, then yes | 20:48 |
| yashgaroth | also centrifuging and resuspending and all that | 20:48 |
| gene_hacker | is it time consuming? | 20:48 |
| kanzure | there's been some people that have done cell culture on a chip.. but meh | 20:49 |
| gene_hacker | or expensive? | 20:49 |
| kanzure | one microbe per droplet, etc. | 20:49 |
| yashgaroth | it's less expensive than PCR | 20:49 |
| yashgaroth | if you want to do anything significant with a plasmid, it'll need to go into bacteria at some point anyway | 20:49 |
| yashgaroth | you can get nanograms from PCR and grams from bacteria | 20:49 |
| kanzure | well your dna that you make doesn't have to be for a plasmid | 20:49 |
| yashgaroth | true | 20:50 |
| kanzure | i think this is definitely down-stream of the dna synthesizer | 20:50 |
| yashgaroth | ok yes fair enough | 20:50 |
| gene_hacker | though the process of joining strands together is PCR correct? | 20:51 |
| yashgaroth | but the sequencing is moot; 99% of anything you read will be normal and block out anything else | 20:51 |
| yashgaroth | no that's ligation | 20:51 |
| gene_hacker | it requires enzymes right? | 20:51 |
| yashgaroth | ligase | 20:51 |
| yashgaroth | you just ligate all your fragments (of whatever size) together, transform into bacteria, and isolate a single clone | 20:51 |
| yashgaroth | otherwise you can't ever be sure all your sequences are correct anyway | 20:52 |
| gene_hacker | so for each synthesis step ligase enzyme would be required | 20:52 |
| yashgaroth | not oligo synthesis | 20:52 |
| yashgaroth | but for assembling oligos together, yes | 20:53 |
| gene_hacker | though I guess if we're working with nanoliters, cost should be minimal | 20:53 |
| yashgaroth | the volume is irrelevant, hell more volume/reagents might cost less than tiny valves and shit | 20:53 |
| yashgaroth | BRB 10 mins, potatoes | 20:54 |
| gene_hacker | so at the very least it does appear somewhat feasible | 20:59 |
| gene_hacker | though it might be good to runs some numbers on error rate and reagent consumption | 21:02 |
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| gene_hacker | though I wonder if nanoliter reactions like this are even feasible? | 21:03 |
| gene_hacker | also looking for a good way to scan a book? | 21:06 |
| kanzure | yes, nanoliters are doable | 21:07 |
| kanzure | good way to scan a book- odesk. | 21:07 |
| kanzure | or elancer | 21:08 |
| kanzure | hire someone in india. mail them the book. give them $30. | 21:08 |
| gene_hacker | can't ship the book to india | 21:08 |
| gene_hacker | well if nanoliters are doable | 21:10 |
| kanzure | nanoliters are definitely doable; and in many cases, biochem is faster on this level | 21:11 |
| kanzure | you don't have to heat a whole whopping mL of liquid | 21:11 |
| gene_hacker | and sufficiently accurate, sufficiently sized robots exist for extracting nanoliters from well plates | 21:12 |
| kanzure | are there well plates that have microfluidic channels built in? | 21:12 |
| gene_hacker | then this shouldn't be too hard to do | 21:12 |
| gene_hacker | no | 21:12 |
| gene_hacker | as long as 1024 different building blocks can be obtained | 21:13 |
| gene_hacker | there are not | 21:13 |
| gene_hacker | plus in order to do something like that you'd need 1024 valves over a very large area | 21:14 |
| gene_hacker | if you move the valve to the well plate then you only need one | 21:14 |
| kanzure | gene_hacker: could i recruit you into this? | 21:14 |
| gene_hacker | possibly | 21:14 |
| gene_hacker | I'd like to see that we can get 1024 different building blocks first or at least a demonstration with a couple | 21:15 |
| gene_hacker | I don't really have that much experience in microfluidics | 21:16 |
| kanzure | btw, did you see delinquentme's liquid handling robot? | 21:17 |
| kanzure | instrument | 21:17 |
| yashgaroth | link please | 21:17 |
| kanzure | http://www.youtube.com/watch?v=ZY5IY5CZ1es | 21:17 |
| kanzure | https://github.com/delinquentme/LH001 | 21:17 |
| kanzure | https://github.com/delinquentme/LH002 | 21:17 |
| delinquentme | ^_^ https://github.com/delinquentme/LH002/raw/master/images/LH002_full_01.png | 21:17 |
| delinquentme | 02 03 04 05 etc | 21:17 |
| delinquentme | kanzure, who do we have who are life extension computational biologists? | 21:18 |
| delinquentme | im about to hit up michael rae to see who he knows | 21:18 |
| yashgaroth | I thought michael rae starved to death | 21:18 |
| delinquentme | O_o;;; | 21:19 |
| yashgaroth | I saw him...3? years ago, he was more emaciated than most cancer patients I've seen | 21:19 |
| yashgaroth | caloric restriction | 21:19 |
| kanzure | computational biologists in life extension.. hrmmm | 21:20 |
| gene_hacker | any work on how long that robot last until needing maintenance? | 21:20 |
| kanzure | ask steve coles | 21:20 |
| kanzure | about the computational biology thing | 21:20 |
| kanzure | ask delinquentme about the maintenance thing | 21:20 |
| gene_hacker | delinquentme any idea how long that thing will last until needing maintenance? | 21:21 |
| gene_hacker | and any metrics of positional accuracy, repeatibility, etc? | 21:22 |
| gene_hacker | also in the early stages of something like this we just need to get a few key parts working | 21:23 |
| delinquentme | gene_hacker, i havnt prototyped it | 21:23 |
| gene_hacker | like high speed ligation and what not | 21:23 |
| delinquentme | steve coles? got an email? | 21:24 |
| gene_hacker | any estimates? | 21:24 |
| kanzure | gene_hacker: sure. | 21:24 |
| kanzure | i think working on this one-part-at-a-time is important | 21:24 |
| kanzure | otherwise it will all break simultaneously | 21:24 |
| gene_hacker | I mean the important thing is the ligation and washing chip, and the well extraction to chip device | 21:25 |
| delinquentme | gene_hacker, So really it should have started out as a smaller project ... could have monetized quicker | 21:25 |
| kanzure | a macroworld well-to-chip would be too slow, i think | 21:25 |
| gene_hacker | and the 1024 different blocks | 21:25 |
| kanzure | maybe ot | 21:25 |
| kanzure | *maybe not | 21:25 |
| gene_hacker | well ligation takes about 30 seconds right? | 21:26 |
| yashgaroth | haha no | 21:26 |
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| kanzure | it depends on reaction volume | 21:26 |
| kanzure | and many other variables | 21:26 |
| kanzure | but i think you can get it down to a few seconds if you are very careful | 21:26 |
| yashgaroth | I'd leave that as long as possible really | 21:27 |
| yashgaroth | at least like 5 minutes | 21:27 |
| delinquentme | kanzure, <3 http://en.wikipedia.org/wiki/L._Stephen_Coles | 21:27 |
| delinquentme | gene_hacker, apologies im not sure if i answered all your questions .. theres a lot going on here | 21:27 |
| gene_hacker | real world to chip is slow, but a lot easier to do than 1024 individually addressable microvalves | 21:28 |
| gene_hacker | I get the hang of it | 21:28 |
| gene_hacker | and after all, you're going for accuracy not speed | 21:29 |
| kanzure | yes | 21:29 |
| yashgaroth | speed is the first thing you'll be sacrificing if you don't have accuracy and money to spare, which I doubt we do | 21:29 |
| kanzure | ehh there's some money to spare | 21:30 |
| yashgaroth | if it's all computerized just run it overnight | 21:30 |
| kanzure | yep | 21:30 |
| gene_hacker | also making a 2 wide microfluidic chip with 1024 vlaves at the LEAST, is insane | 21:30 |
| gene_hacker | in reality, we'd probably need much more than that to do multiplexing and what not. | 21:32 |
| gene_hacker | even with droplet based microfluidics it's insane | 21:32 |
| delinquentme | i like the part where Ghost in the shell | 21:40 |
| kanzure | delinquentme: ? | 21:43 |
| delinquentme | its a solid anime :D | 21:43 |
| kanzure | are you new to anime | 21:44 |
| * SDr mumbles incomprehensible about Serial Experiments Lain | 21:45 | |
| delinquentme | haha not really but i wouldnt say im well versed | 21:46 |
| delinquentme | like Neon Genesis Evangelion is hot shit :D | 21:46 |
| kanzure | this moment brought to you by japan | 21:46 |
| kanzure | http://www.youtube.com/watch?v=6AYaFL6SWmk#t=1m40s | 21:46 |
| delinquentme | OMG ROBOT WANT | 21:46 |
| kanzure | SDr: that one was nice.. | 21:46 |
| delinquentme | lolol watching a thing on nat geo about hallucinogens | 21:48 |
| delinquentme | total straight up white middle texas guy taking shrooms | 21:48 |
| delinquentme | " Eets Lyke the WHitest WHHite" | 21:48 |
| delinquentme | donloading lain :D | 21:49 |
| delinquentme | lololol | 21:49 |
| delinquentme | nice vid kanzure | 21:49 |
| delinquentme | kanzure, have you ever tried to contact ben langmead of crossbow fame? | 21:50 |
| kanzure | nope haven't talked with him | 21:51 |
| delinquentme | hadoop or disco | 21:52 |
| delinquentme | aka java or erlang | 21:52 |
| delinquentme | well java erlang python | 21:52 |
| delinquentme | i might start programming deh java | 21:52 |
| kanzure | java isn't hard to figure out | 21:53 |
| delinquentme | I've programmed in it in HS | 21:56 |
| delinquentme | looking for something just FAST :D | 21:56 |
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| delinquentme | is there a term for timing your large processing jobs in an API as to not flood them w requests? | 22:24 |
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| kanzure | delinquentme: queuing? | 22:27 |
| kanzure | look up aws sqs | 22:27 |
| kanzure | or rabbitmq and shit | 22:28 |
| delinquentme | throttling is what I was aftar | 22:28 |
| kanzure | just spend more money throwing up new instances :P | 22:29 |
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