Updated Uncalled and Minknow No Longer Working

[sbabbbie]

Hello,

I am a researcher working on enriching fungal pathogen genomes from mammal host tissues, which I have successfully done using your UNCALLED software in the past. However, after being forced to update MinKNOW and then updating UNCALLED so it would work with the newest version, UNCALLED has essentially stopped functioning.

In the .paf files, no reads are being kept even though I know there is target DNA in the sample, and mapping to the target afterward using minimap2 does show there are reads from the target. Additionally, although the .paf says that reads are being ejected, I don't see the characteristic "spike" in the read length distribution in MinKNOW at the length they say they are being ejected at. The size distribution is in fact identical to a run I performed without UNCALLED, suggesting to me that UNCALLED is simply not working or actually enriching for my target.

Do you have any guidance?

Thank you!

[skovaka]

Very sorry for the trouble, I'm working on getting a sequencing run together so I can look into this. It is probably an issue with the new MinKNOW version. I recently tested the MinKNOW connection on an old flowcell (#44), but did not confirm that reads were being properly ejected.

I will hopefully be able to check on this within the next week, but in the meantime you could try using ReadFish, or MinKNOW's builtin "adaptive sampling" method

[sbabbbie]

Hello Sam, Thank you so much for the quick response! I am also going to try out Uncalled on a sample I have run before and I know to have high amounts of the target DNA to confirm the replicability of the issue within the next few days, and will report back. Looking forward to hearing back from you. Spenser Babb-Biernacki Pronouns: she/her/hers PhD Student, Louisiana State University ***@***.*** | 623-210-3782

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________________________________ From: Sam Kovaka ***@***.***> Sent: Thursday, June 23, 2022 1:15:42 PM To: skovaka/UNCALLED ***@***.***> Cc: Spenser Biernacki ***@***.***>; Author ***@***.***> Subject: Re: [skovaka/UNCALLED] Updated Uncalled and Minknow No Longer Working (Issue #46) You don't often get email from ***@***.*** Learn why this is important<https://aka.ms/LearnAboutSenderIdentification> Very sorry for the trouble, I'm working on getting a sequencing run together so I can look into this. It is probably an issue with the new MinKNOW version. I recently tested the MinKNOW connection on an old flowcell (#44<https://nam04.safelinks.protection.outlook.com/?url=https%3A%2F%2Fgithub.com%2Fskovaka%2FUNCALLED%2Fissues%2F44&data=05%7C01%7Csbiern1%40lsu.edu%7Cac21c9f532be42dfdb1d08da55446299%7C2d4dad3f50ae47d983a09ae2b1f466f8%7C0%7C0%7C637916049443576870%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000%7C%7C%7C&sdata=%2BVQGn%2BYke%2BCqzqTzkjJ%2Fj3mcp7VtyZWCqjuPsSqRVbM%3D&reserved=0>), but did not confirm that reads were being properly ejected. I will hopefully be able to check on this within the next week, but in the meantime you could try using ReadFish<https://nam04.safelinks.protection.outlook.com/?url=https%3A%2F%2Fgithub.com%2FLooseLab%2Freadfish&data=05%7C01%7Csbiern1%40lsu.edu%7Cac21c9f532be42dfdb1d08da55446299%7C2d4dad3f50ae47d983a09ae2b1f466f8%7C0%7C0%7C637916049443576870%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000%7C%7C%7C&sdata=W6vY1Y0YxKvEG4IthYD6dYfqzHaL1Ik6sqPQXsuk0Yk%3D&reserved=0>, or MinKNOW's builtin "adaptive sampling" method — Reply to this email directly, view it on GitHub<https://nam04.safelinks.protection.outlook.com/?url=https%3A%2F%2Fgithub.com%2Fskovaka%2FUNCALLED%2Fissues%2F46%23issuecomment-1164724641&data=05%7C01%7Csbiern1%40lsu.edu%7Cac21c9f532be42dfdb1d08da55446299%7C2d4dad3f50ae47d983a09ae2b1f466f8%7C0%7C0%7C637916049443576870%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000%7C%7C%7C&sdata=OfBbDt83PegyeAKnptElCgv763teTUEKxN7tr6c%2B8jI%3D&reserved=0>, or unsubscribe<https://nam04.safelinks.protection.outlook.com/?url=https%3A%2F%2Fgithub.com%2Fnotifications%2Funsubscribe-auth%2FALDLSDNMVP66ULID4O3GZIDVQSSU5ANCNFSM5ZRWXKPQ&data=05%7C01%7Csbiern1%40lsu.edu%7Cac21c9f532be42dfdb1d08da55446299%7C2d4dad3f50ae47d983a09ae2b1f466f8%7C0%7C0%7C637916049443576870%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000%7C%7C%7C&sdata=nxM8lNHbIMUsmMIE4q5Wm8q9PZKZTVIkqROiiRtkHd0%3D&reserved=0>. You are receiving this because you authored the thread.Message ID: ***@***.***>

[skovaka]

Sorry for the delay, I have identified and fixed the main issue, which was related to signal calibration which MinKNOW used to perform but no longer does. Unfortunately, the ReadUntil API appears to be more finicky in general with the new update. I found that briefly running MinKNOW's builtin adaptive sampling fixes the problem. I've added a warning to the README which describes the problem and solution in more detail: https://github.com/skovaka/UNCALLED#real-time-readuntil

I know that's not an ideal solution, I will try to look into it further in the future.

Thanks,
Sam

[sbabbbie]

Hi Sam, Thank you so much for taking the time to work on this issue. I’m out of town for the next week, but when I get back I will do a brief trial run to see if the issue is resolved. Cheers, Spenser

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________________________________ From: Sam Kovaka ***@***.***> Sent: Thursday, July 7, 2022 2:18:11 PM To: skovaka/UNCALLED ***@***.***> Cc: Spenser Biernacki ***@***.***>; Author ***@***.***> Subject: Re: [skovaka/UNCALLED] Updated Uncalled and Minknow No Longer Working (Issue #46) You don't often get email from ***@***.*** Learn why this is important<https://aka.ms/LearnAboutSenderIdentification> Sorry for the delay, I have identified and fixed the main issue, which was related to signal calibration which MinKNOW used to perform but no longer does. Unfortunately, the ReadUntil API appears to be more finicky in general with the new update. I found that briefly running MinKNOW's builtin adaptive sampling fixes the problem. I've added a warning to the README which describes the problem and solution in more detail: https://github.com/skovaka/UNCALLED/edit/master/README.md#real-time-readuntil<https://nam04.safelinks.protection.outlook.com/?url=https%3A%2F%2Fgithub.com%2Fskovaka%2FUNCALLED%2Fedit%2Fmaster%2FREADME.md%23real-time-readuntil&data=05%7C01%7Csbiern1%40lsu.edu%7Cb35f7d1023fe4330f71908da604d6f54%7C2d4dad3f50ae47d983a09ae2b1f466f8%7C0%7C0%7C637928182939868423%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000%7C%7C%7C&sdata=v0iWIcWB151Fd1VCeHvrFzNo79p3zU1Rm52LOTXPaMk%3D&reserved=0> I know that's not an ideal solution, I will try to look into it further in the future. Thanks, Sam — Reply to this email directly, view it on GitHub<https://nam04.safelinks.protection.outlook.com/?url=https%3A%2F%2Fgithub.com%2Fskovaka%2FUNCALLED%2Fissues%2F46%23issuecomment-1178110555&data=05%7C01%7Csbiern1%40lsu.edu%7Cb35f7d1023fe4330f71908da604d6f54%7C2d4dad3f50ae47d983a09ae2b1f466f8%7C0%7C0%7C637928182939868423%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000%7C%7C%7C&sdata=W7gvyKHXasWNVVh%2B1%2FgWQ7f27eY65PuLDLdPzcIkHUM%3D&reserved=0>, or unsubscribe<https://nam04.safelinks.protection.outlook.com/?url=https%3A%2F%2Fgithub.com%2Fnotifications%2Funsubscribe-auth%2FALDLSDPCHO7D7TEKG7XX4F3VS4UPHANCNFSM5ZRWXKPQ&data=05%7C01%7Csbiern1%40lsu.edu%7Cb35f7d1023fe4330f71908da604d6f54%7C2d4dad3f50ae47d983a09ae2b1f466f8%7C0%7C0%7C637928182939868423%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000%7C%7C%7C&sdata=vM3wf5VgCKTykpyFFzyvdv%2F4Fq2h1bBGXxXvgb%2BQg2c%3D&reserved=0>. You are receiving this because you authored the thread.Message ID: ***@***.***>