notes - Zymo Research HostZero Microbial DNA Kit (used for shotgun sequencing) - 16S error correction tools: > Divisive Amplicon Denoising Algorithm 2 or DADA2, an open-source R package that improves on the DADA algorithm > Quantitative Insights Into Microbial Ecology or QIIME (canonically pronounced 'Chime') - 16s bioinformatic tool: PICRUSt (pronounced “pie crust”) or Phylogenetic Investigation of Communities by Reconstruction of Unobserved States, written in Python and R Thanks for this wonderful video. 🌞
@Kevin-ox7zp3 ай бұрын
How do i store the filtered dna?
@ZymoResearch2 ай бұрын
Hi Kevin, we would recommend storing the purified DNA in either a -20C or -80C freezer to ensure long term stability.
@soniavargas79474 ай бұрын
this was an excellent video, thank you so much! The autocorrect made me laugh, as this has victimized me in the past.
@harshathanreddy7175 ай бұрын
Thank you so much it was really helpful
@ZymoResearch2 ай бұрын
We are so glad the video was helpful!
@Falany6 ай бұрын
For how long should you thaw on ice?
@ZymoResearch2 ай бұрын
For the initial thawing step prior to adding DNA, it usually only takes about 2-3 minutes. We usually recommend flicking the tube with the competent cells just to see if the contents is still solid. If it is, then you can let the tube thaw for a few more minutes.
@TruongTran-qo3sf8 ай бұрын
Thanks for your presentation, what is the tool to find DMR in your research?
@rashamoustafa4279 ай бұрын
Great🎉and thanks
@webstermurwira10 ай бұрын
11122
@aseelaraji704510 ай бұрын
Looking forward to see the results
@kiawendy745 Жыл бұрын
fantastic video, thanksss
@ZymoResearch10 ай бұрын
Glad you liked it!
@magicdiamond8767 Жыл бұрын
If I want to measure Methylation of protocadherin 17 human gene in gastric cancer cells before and after adding of dnmt1 inhibitor what is methods can be used ?
@Knowledgepluggin Жыл бұрын
Can it be stored for a prolonged period of 6months
@ZymoResearch2 ай бұрын
Absolutely! For samples stored in DNA/RNA Shield: - DNA: Stable for up to 2 years at room temperature - RNA: Stable for up to 1 month at room temperature For longer term storage, you can also store the samples at -20C or -80C.
@habeebahtitilola8015 Жыл бұрын
Hello Zymo, Can i use a thermo mixer in place of Bead beater?
@ZymoResearch2 ай бұрын
Hi Habeebah, we would not recommend using a Thermo Mixer for the bead beating step. The Thermo Mixer will just shake the beads, but it won't move them in the right orientation so that they break open the microbial cells. In this case, you most likely will end up with low yields and underrepresentation of harder-to-lyse microbes like gram-positive bacteria or fungi. Here is a list of bead beating instruments and conditions that we would recommend: files.zymoresearch.com/documents/bead_beating_short_protocol_tables.pdf The easiest to set up would be the Vortex Genie from Scientific Industries with a Horizontal Tube Adapter (see links below): www.zymoresearch.com/products/vortex-genie-2 www.zymoresearch.com/products/horizontal-microtube-holder
@ope4422 Жыл бұрын
Tip to increase plasmid yield exponentially: Before using kit, spin down 1mL of culture and discard supernatant. Re-suspend in 1:1 LB and H20, ~600uL. Continue using plasmic miniprep kit as instructed. This technique has increased yield 7x in some cases. Hope this helps someone else!
@CR-pg8bd2 ай бұрын
Thank you for sharing this tip. Sorry if this is a silly question, but why do you recommend resuspending in 1:1 LB and H2O specifically, versus just ddH2O or LB for example?
@davidg.johnson7208 Жыл бұрын
Increase NAD, decrease CD38 and control body inflammation seems to be the way right now to live a long healthy life that the average person can do right now.
@loxix100loxix100 Жыл бұрын
how do we know if the sequence is or isn’t conserved?
@Jackoline6 Жыл бұрын
thank you this is amazing
@maxinecutracci1940 Жыл бұрын
Hi, can I use a power lyzer instead of the bead beater? and for how long? Thanks !
@ZymoResearch2 ай бұрын
Hi Maxine, you can use the PowerLyzer 24 Homogenizer for the bead beating steps. I believe that instrument is very similar to the Omni Bead Ruptor Elite instrument, so you can use those conditions as we've outlined here in our Optimized Lysis Protocols sheet files.zymoresearch.com/documents/bead_beating_short_protocol_tables.pdf:
@ktburger659 Жыл бұрын
Great video, thanks so much
@marilynemawugnon59892 жыл бұрын
Can you also explain to me a bit what do you mean by cross-domain coverage?
@marilynemawugnon59892 жыл бұрын
Hey Zymo Research... pls do you have in store some references discussing about metagenomics vs metabarcoding to share with us (me especially)? Thanks by advance!
@drcaoimhingriffin90482 жыл бұрын
At the initial TRizol/Ethanol mixing step, would using 100% Ethanol instead of 95% Ethanol affect the RNA yield from the extraction
@ZymoResearch2 ай бұрын
Good question! At this step, 95-100% ethanol can be used and the kit performance/RNA yield will be the same. The more important thing is that the ratio remains at 1:1 with the volume of TRIzol.
@muhammadakmal14142 жыл бұрын
This kit can be used for gram positive bacteria plasmid Dna isolation?
@ZymoResearch2 ай бұрын
Yes, there is an appendix procedure for isolating plasmid DNA from Gram-Positive bacteria using the ZymoPURE Plasmid kits. You can find it on pg. 9 of the following Maxiprep protocol (link below). The main modification is to include a lytic enzyme (e.g. lysozyme) to help break down the cell wall of hardier Gram-Positive bacteria. files.zymoresearch.com/protocols/_d4202_d4203_zymopure_ii_plasmid_maxiprep.pdf
@oscarmunoz22692 жыл бұрын
This was a great presentation. Thank you!
@adeelmanzoor98012 жыл бұрын
I think the instructor should slow down as speedy talk make understanding such technical topics more difficult.
@G-gnome2 жыл бұрын
BS sales spiel. Tested negative with rapid and PCR, two days later at an airport tested positive. Came back to retest with a negative. How about a statistical report on how testing sites are following protocols…
@aprilmaetabonda20203 жыл бұрын
Thank you very much for this detailed and excellent presentation. Btw, I used Zymo RNA Concentrator during my extraction :)
@pankajchand67613 жыл бұрын
Why does the speaker need to speak so fast when explaining a technical topic?
@edodanilyan2 жыл бұрын
Well, you can slow it down, there's a feature to do that in KZread
@sakina3023 жыл бұрын
Thank you for the helpful information! Really appreciate it
@charlietango95233 жыл бұрын
How large of a pellet is too large?
@SELFEMPOWERMENTFORWOMEN3 жыл бұрын
I believe I've created an epigenetic emotional and mental psychological tool that's getting 100% results in resolving the cyclic negative behavioural patterns. I built it based on realising you have to work within a bloodline to be sure you get the root. Although I've got hundreds of real people case studies, I don't have anything scientific. I think this is a mahout missing live of the puzzle and it's organic. I also don't have the funding for an official study. Although my tool is now used in 20 countries by therapists. It really is a one of a kind. I think all studies need to also be segmented into males and females. Keep up the great work guys.
@SELFEMPOWERMENTFORWOMEN3 жыл бұрын
The results are based on a pre screening 'rightness and readiness' of course to ensure they completely do their part.
@speicaldark3 жыл бұрын
This is a really nice video. Very helpful!
@asbestosflake57493 жыл бұрын
Great video. I have a question. In the first scenario it was around a 9% chance of a false positive and an almost 0% chance of a false negative. Do labs do any additional calculations to combine the two (positives and negative numbers) to give a more accurate result? Basically I’m trying to work out in the first scenario if the person got a positive result then does that mean it’s 91% accurate or is it more accurate than that when you factor in the negative numbers?? Hope that makes sense…
@prakroothi3 жыл бұрын
Hi, Could you please tell me how deduplication of the alignment was done using Bismark ?
@ZymoResearch3 жыл бұрын
Hi, Thank you for your inquiry. Our Technical Support team would be more than happy to assist you or answer any of your questions, please send us an email to [email protected] and someone will get back to you shortly.
@jacksonhuang89523 жыл бұрын
Is the strong smell during the preparation harmful to us?
@ZymoResearch3 жыл бұрын
Hello, the strong fumes are usually from the TRIzol reagent. In low amounts, the fumes are not harmful. However, we recommend performing the sample lysis and loading step in a fume hood to minimize the amount of exposure to the fumes.
@nidhisukhija37523 жыл бұрын
How to index reference genome using bismark....please share the command usage
@aprilmaetabonda20203 жыл бұрын
Is this feasible for RNA purification?
@claudiazelazny76813 жыл бұрын
Would the blood transfusion of which you spoke have a positive outcome for infertility cases?
@absdell53813 жыл бұрын
Really helpful sir. Thanks a lot.
@ETalaz3 жыл бұрын
Is it that hard to put subtitle really?
@danielgladish25023 жыл бұрын
Great video! A suggestion for something you could do in the future might be to make a video comparing Amplicon Sequencing and Shotgun Sequencing, as 16S is just an example of an amplicon. You state that the use of 16S region limits your study to just bacteria, but other highly conserved regions can be used to target members of the other domains, like the 18S and ITS regions like you mentioned at the end.
@marilynemawugnon59892 жыл бұрын
Good point
@Crazy09starkillor3 жыл бұрын
If i were to use ASV, do I still need to run DADA2 or amplicon denoise before hand?
@couchpatata68358 ай бұрын
When you run your sequences using DADA2 (I used the DADA2 plugin in QIIME2), your output are ASVs.
@Crazy09starkillor3 жыл бұрын
Why are there no error correction tools for shotgun sequencing?
@Bob_Adkins2 жыл бұрын
My guess is that the tests go through a correction algorithm, and are already factored in.
@alfianabdulhalin18733 жыл бұрын
Thanks for the video Doc :) A good explanation. I have a question though. From what I understand, at the current moment... we cannot know whether someone has covid-19 without doing the RT-PCR test, right? ... One can only know whether they are infected only based on this test. The thing is... when people talk about FP and FN (false positives and negatives) ... one thing that is required is the actual number of positive (true positive) people and also the actual number of those without the disease (true negative).... Now... since we don't have any true positive numbers (since the RT-PCR itself is not a gold standard)... how can FP or any other metric (e.g. sensitivity, specificity, or even FN) be reported or calculated? Ideally (or supposedly), before the RT-PCR would even be administered... one would have to have a sure way of knowing whether a person has the disease or not. Say this is done through isolating and purifying test subjects samples and absolutely determining whether or not he/she has the virus. In short, this is the gold standard... where the ground truth is known (meaning we know who actually has the virus, and who does not have the virus). Then, after these subjects have been properly classified.... then we run the RT-PCR on them... to see which patients are classified as +ve and which as -ve. .... and only then can we calculate the false positive (or whatever other) metric, right? Now... with the absenece of such subjects... how can we say that the RT-PCR has a FP of such and such? Or an FN of such and such....? Please note that this is not a negative comment. I am just trying to clarify based on what I've learned in my research on how to calculate false positives, sensitivity etc. :) Thank you!
@immaculatesquid3 жыл бұрын
if you're talking about Covid-19 specifically, the PCR tests according to the CDC, identify coronaviruses, not necessarily covid-19. This could lead to a quite high false positive rate.
@kianaliu66363 жыл бұрын
Dear Partner, It’s my honor to meet you here. This is Kiana from China, I work for a localization company with 120 languages in Asia I see that your company's business involves multiple continents. Do you need localization and translation services? May I get some Translation or localization projects from your company? I am willing to translate the 300-word document for you free of charge to prove the quality. Email: [email protected] WhatsApp / Tel:86-18319523416
@yadansong62133 жыл бұрын
Could you introduce how to analyze differential methylation when you are free? And the theoroy about how to get methylation signal as well as interpret it in detail .
@biokmst3 жыл бұрын
This doesn't explain the tests ability to recognize any coronavirus antibodies that have resulted from The Common Cold. You need to add that into your data to increase the odds of a false positive including a biological aspect. See CDC website regarding.
@tenton2000k3 жыл бұрын
This video has nothing to do with that. It just goes over how statistical results are generated.
@BiologyIsHot4 жыл бұрын
Curious how cheap I could drive sequencing for some very low complexity population samples. I'm interested in getting full genomes for some samples that probably have 10 or less species/strains as cheap as possible.
@aaronsilver-pell4114 жыл бұрын
This is fantastic thank you folks so much!
@user-wy4rx6fp8u4 жыл бұрын
Thanks for uploading ~ Great video, it provides an informative insight into the two methods.
Пікірлер
notes - Zymo Research HostZero Microbial DNA Kit (used for shotgun sequencing) - 16S error correction tools: > Divisive Amplicon Denoising Algorithm 2 or DADA2, an open-source R package that improves on the DADA algorithm > Quantitative Insights Into Microbial Ecology or QIIME (canonically pronounced 'Chime') - 16s bioinformatic tool: PICRUSt (pronounced “pie crust”) or Phylogenetic Investigation of Communities by Reconstruction of Unobserved States, written in Python and R Thanks for this wonderful video. 🌞
How do i store the filtered dna?
Hi Kevin, we would recommend storing the purified DNA in either a -20C or -80C freezer to ensure long term stability.
this was an excellent video, thank you so much! The autocorrect made me laugh, as this has victimized me in the past.
Thank you so much it was really helpful
We are so glad the video was helpful!
For how long should you thaw on ice?
For the initial thawing step prior to adding DNA, it usually only takes about 2-3 minutes. We usually recommend flicking the tube with the competent cells just to see if the contents is still solid. If it is, then you can let the tube thaw for a few more minutes.
Thanks for your presentation, what is the tool to find DMR in your research?
Great🎉and thanks
11122
Looking forward to see the results
fantastic video, thanksss
Glad you liked it!
If I want to measure Methylation of protocadherin 17 human gene in gastric cancer cells before and after adding of dnmt1 inhibitor what is methods can be used ?
Can it be stored for a prolonged period of 6months
Absolutely! For samples stored in DNA/RNA Shield: - DNA: Stable for up to 2 years at room temperature - RNA: Stable for up to 1 month at room temperature For longer term storage, you can also store the samples at -20C or -80C.
Hello Zymo, Can i use a thermo mixer in place of Bead beater?
Hi Habeebah, we would not recommend using a Thermo Mixer for the bead beating step. The Thermo Mixer will just shake the beads, but it won't move them in the right orientation so that they break open the microbial cells. In this case, you most likely will end up with low yields and underrepresentation of harder-to-lyse microbes like gram-positive bacteria or fungi. Here is a list of bead beating instruments and conditions that we would recommend: files.zymoresearch.com/documents/bead_beating_short_protocol_tables.pdf The easiest to set up would be the Vortex Genie from Scientific Industries with a Horizontal Tube Adapter (see links below): www.zymoresearch.com/products/vortex-genie-2 www.zymoresearch.com/products/horizontal-microtube-holder
Tip to increase plasmid yield exponentially: Before using kit, spin down 1mL of culture and discard supernatant. Re-suspend in 1:1 LB and H20, ~600uL. Continue using plasmic miniprep kit as instructed. This technique has increased yield 7x in some cases. Hope this helps someone else!
Thank you for sharing this tip. Sorry if this is a silly question, but why do you recommend resuspending in 1:1 LB and H2O specifically, versus just ddH2O or LB for example?
Increase NAD, decrease CD38 and control body inflammation seems to be the way right now to live a long healthy life that the average person can do right now.
how do we know if the sequence is or isn’t conserved?
thank you this is amazing
Hi, can I use a power lyzer instead of the bead beater? and for how long? Thanks !
Hi Maxine, you can use the PowerLyzer 24 Homogenizer for the bead beating steps. I believe that instrument is very similar to the Omni Bead Ruptor Elite instrument, so you can use those conditions as we've outlined here in our Optimized Lysis Protocols sheet files.zymoresearch.com/documents/bead_beating_short_protocol_tables.pdf:
Great video, thanks so much
Can you also explain to me a bit what do you mean by cross-domain coverage?
Hey Zymo Research... pls do you have in store some references discussing about metagenomics vs metabarcoding to share with us (me especially)? Thanks by advance!
At the initial TRizol/Ethanol mixing step, would using 100% Ethanol instead of 95% Ethanol affect the RNA yield from the extraction
Good question! At this step, 95-100% ethanol can be used and the kit performance/RNA yield will be the same. The more important thing is that the ratio remains at 1:1 with the volume of TRIzol.
This kit can be used for gram positive bacteria plasmid Dna isolation?
Yes, there is an appendix procedure for isolating plasmid DNA from Gram-Positive bacteria using the ZymoPURE Plasmid kits. You can find it on pg. 9 of the following Maxiprep protocol (link below). The main modification is to include a lytic enzyme (e.g. lysozyme) to help break down the cell wall of hardier Gram-Positive bacteria. files.zymoresearch.com/protocols/_d4202_d4203_zymopure_ii_plasmid_maxiprep.pdf
This was a great presentation. Thank you!
I think the instructor should slow down as speedy talk make understanding such technical topics more difficult.
BS sales spiel. Tested negative with rapid and PCR, two days later at an airport tested positive. Came back to retest with a negative. How about a statistical report on how testing sites are following protocols…
Thank you very much for this detailed and excellent presentation. Btw, I used Zymo RNA Concentrator during my extraction :)
Why does the speaker need to speak so fast when explaining a technical topic?
Well, you can slow it down, there's a feature to do that in KZread
Thank you for the helpful information! Really appreciate it
How large of a pellet is too large?
I believe I've created an epigenetic emotional and mental psychological tool that's getting 100% results in resolving the cyclic negative behavioural patterns. I built it based on realising you have to work within a bloodline to be sure you get the root. Although I've got hundreds of real people case studies, I don't have anything scientific. I think this is a mahout missing live of the puzzle and it's organic. I also don't have the funding for an official study. Although my tool is now used in 20 countries by therapists. It really is a one of a kind. I think all studies need to also be segmented into males and females. Keep up the great work guys.
The results are based on a pre screening 'rightness and readiness' of course to ensure they completely do their part.
This is a really nice video. Very helpful!
Great video. I have a question. In the first scenario it was around a 9% chance of a false positive and an almost 0% chance of a false negative. Do labs do any additional calculations to combine the two (positives and negative numbers) to give a more accurate result? Basically I’m trying to work out in the first scenario if the person got a positive result then does that mean it’s 91% accurate or is it more accurate than that when you factor in the negative numbers?? Hope that makes sense…
Hi, Could you please tell me how deduplication of the alignment was done using Bismark ?
Hi, Thank you for your inquiry. Our Technical Support team would be more than happy to assist you or answer any of your questions, please send us an email to [email protected] and someone will get back to you shortly.
Is the strong smell during the preparation harmful to us?
Hello, the strong fumes are usually from the TRIzol reagent. In low amounts, the fumes are not harmful. However, we recommend performing the sample lysis and loading step in a fume hood to minimize the amount of exposure to the fumes.
How to index reference genome using bismark....please share the command usage
Is this feasible for RNA purification?
Would the blood transfusion of which you spoke have a positive outcome for infertility cases?
Really helpful sir. Thanks a lot.
Is it that hard to put subtitle really?
Great video! A suggestion for something you could do in the future might be to make a video comparing Amplicon Sequencing and Shotgun Sequencing, as 16S is just an example of an amplicon. You state that the use of 16S region limits your study to just bacteria, but other highly conserved regions can be used to target members of the other domains, like the 18S and ITS regions like you mentioned at the end.
Good point
If i were to use ASV, do I still need to run DADA2 or amplicon denoise before hand?
When you run your sequences using DADA2 (I used the DADA2 plugin in QIIME2), your output are ASVs.
Why are there no error correction tools for shotgun sequencing?
My guess is that the tests go through a correction algorithm, and are already factored in.
Thanks for the video Doc :) A good explanation. I have a question though. From what I understand, at the current moment... we cannot know whether someone has covid-19 without doing the RT-PCR test, right? ... One can only know whether they are infected only based on this test. The thing is... when people talk about FP and FN (false positives and negatives) ... one thing that is required is the actual number of positive (true positive) people and also the actual number of those without the disease (true negative).... Now... since we don't have any true positive numbers (since the RT-PCR itself is not a gold standard)... how can FP or any other metric (e.g. sensitivity, specificity, or even FN) be reported or calculated? Ideally (or supposedly), before the RT-PCR would even be administered... one would have to have a sure way of knowing whether a person has the disease or not. Say this is done through isolating and purifying test subjects samples and absolutely determining whether or not he/she has the virus. In short, this is the gold standard... where the ground truth is known (meaning we know who actually has the virus, and who does not have the virus). Then, after these subjects have been properly classified.... then we run the RT-PCR on them... to see which patients are classified as +ve and which as -ve. .... and only then can we calculate the false positive (or whatever other) metric, right? Now... with the absenece of such subjects... how can we say that the RT-PCR has a FP of such and such? Or an FN of such and such....? Please note that this is not a negative comment. I am just trying to clarify based on what I've learned in my research on how to calculate false positives, sensitivity etc. :) Thank you!
if you're talking about Covid-19 specifically, the PCR tests according to the CDC, identify coronaviruses, not necessarily covid-19. This could lead to a quite high false positive rate.
Dear Partner, It’s my honor to meet you here. This is Kiana from China, I work for a localization company with 120 languages in Asia I see that your company's business involves multiple continents. Do you need localization and translation services? May I get some Translation or localization projects from your company? I am willing to translate the 300-word document for you free of charge to prove the quality. Email: [email protected] WhatsApp / Tel:86-18319523416
Could you introduce how to analyze differential methylation when you are free? And the theoroy about how to get methylation signal as well as interpret it in detail .
This doesn't explain the tests ability to recognize any coronavirus antibodies that have resulted from The Common Cold. You need to add that into your data to increase the odds of a false positive including a biological aspect. See CDC website regarding.
This video has nothing to do with that. It just goes over how statistical results are generated.
Curious how cheap I could drive sequencing for some very low complexity population samples. I'm interested in getting full genomes for some samples that probably have 10 or less species/strains as cheap as possible.
This is fantastic thank you folks so much!
Thanks for uploading ~ Great video, it provides an informative insight into the two methods.