Setting reading frame for "Amino Acid plot for" amplicon

[crubinstein]

Hi All,

I like the new amino acid quilt that illustrates the translations for all unique alleles. It seems the default reading frame to translate conding_seq is 0. I imagine there must be a way to set this? Is there, and what is the parameter name?

Thanks!

Dustin

[Colelyman]

Hey Dustin,

Thanks for using CRISPResso, and glad to hear you like the new amino acid quilt! When we developed it, we assumed that the reading frame would start at the beginning of coding_seq, so we currently don't have a way to set this. Do you have a use case where your reading frame doesn't start at the beginning of coding_seq? (I assume that you do because you are asking the question). Also, just want to clarify, your coding_seq is within (i.e. smaller than) your amplicon_seq, right?

Thanks,
Cole

[crubinstein]

Hi Cole, Thanks for your work. I’ve been a long time user of CRISPResso for a while now. Yes, coding_seq is fully contained in amplicon_seq. I would say that on average, the start of the coding sequence is in the 0 frame about only 33% of the time. I would imagine these CDS are most always exon boundaries in practice. And of course, exons can begin in any of 3 reading frames (FWIW, you might also consider the negative strand reading frames, as sometimes my amplicon sequence might be antisense to the CDS). Just a suggestion that I could imagine a reading frame parameter (with possible values +0,+1,+2,-0,-1,-2) that could be comma delimited with same number of values as coding_seq. * I know the +0/-0 is, aesthetically, a little unsightly, but I think that’s the most standard way to reference reading frames. Imagine it as the number of bases that need to be removed before you can begin assessing codons. * In most cases (in my experience), multiple, comma-delimited coding_seq values are consecutive exons. In those cases, the coding sequence continues from the previous, so you wouldn’t need to specifiy the reading frame of each coding_seq. I suppose I’m proposing a user could provide (1) a reading_frame with the same number of values as coding_seq or (2) a single reading_frame value, and all reading frames stem from that (essentially you would just concatenate all coding_seq values together and apply the single reading_frame). CRISPResso2’s behavior would be dependent on the number of values in coding_seq. Thanks, Dustin

…

-- C. Dustin Rubinstein, PhD (he/him) Director, Advanced Genome Editing Laboratory University of Wisconsin Biotechnology Center Genetics-Biotech Building, Rm 3122 608.890.1507 • ***@***.*** Ho-Chunk Land Acknowledgement<https://compliance.wisc.edu/land-acknowledgement/> From: Cole Lyman ***@***.***> Date: Tuesday, April 28, 2026 at 6:22 PM To: pinellolab/CRISPResso2 ***@***.***> Cc: C. Dustin Rubinstein ***@***.***>; Author ***@***.***> Subject: Re: [pinellolab/CRISPResso2] Setting reading frame for "Amino Acid plot for" amplicon (Discussion #642) Hey Dustin, Thanks for using CRISPResso, and glad to hear you like the new amino acid quilt! When we developed it, we assumed that the reading frame would start at the beginning of coding_seq, so we currently don't have a way to set this. Do you have a use case where your reading frame doesn't start at the beginning of coding_seq? (I assume that you do because you are asking the question). Also, just want to clarify, your coding_seq is within (i.e. smaller than) your amplicon_seq, right? Thanks, Cole — Reply to this email directly, view it on GitHub<https://urldefense.com/v3/__https://github.com/pinellolab/CRISPResso2/discussions/642*discussioncomment-16749091__;Iw!!Mak6IKo!JZj4oNFfc4AlQejfTPWieWWA8PUL_68NJczf62fNDKXS1VP_2cul2CQLdenwpXDvQUl9OgkTHOB_hv6INbae9Z-NXO4$>, or unsubscribe<https://urldefense.com/v3/__https://github.com/notifications/unsubscribe-auth/BHVZEPZXLHANP6LK3HAGK234YE4MHAVCNFSM6AAAAACYGFDCAWVHI2DSMVQWIX3LMV43URDJONRXK43TNFXW4Q3PNVWWK3TUHMYTMNZUHEYDSMI__;!!Mak6IKo!JZj4oNFfc4AlQejfTPWieWWA8PUL_68NJczf62fNDKXS1VP_2cul2CQLdenwpXDvQUl9OgkTHOB_hv6INbaehwbf4nM$>. Triage notifications on the go with GitHub Mobile for iOS<https://urldefense.com/v3/__https://apps.apple.com/app/apple-store/id1477376905?ct=notification-email&mt=8&pt=524675__;!!Mak6IKo!JZj4oNFfc4AlQejfTPWieWWA8PUL_68NJczf62fNDKXS1VP_2cul2CQLdenwpXDvQUl9OgkTHOB_hv6INbaeCw56KWo$> or Android<https://urldefense.com/v3/__https://play.google.com/store/apps/details?id=com.github.android&referrer=utm_campaign*3Dnotification-email*26utm_medium*3Demail*26utm_source*3Dgithub__;JSUlJSU!!Mak6IKo!JZj4oNFfc4AlQejfTPWieWWA8PUL_68NJczf62fNDKXS1VP_2cul2CQLdenwpXDvQUl9OgkTHOB_hv6INbaeZaTS0x8$>. You are receiving this because you authored the thread.

[Colelyman]

Hi Dustin,

Thanks for the explanation, for now we will support this by recommending that you set your coding_seq to be the start of the reading frame, and supplying the RC of the amplicon to support coding_seqs that may be antisense to the amplicon. The main motivation behind this decision is to try to keep complexity low (and not add yet another parameter to CRISPResso).

Thank you for the feedback!
Cole

[crubinstein]

Hi Cole, Actually, I fully understand the need to keep complexity low. I don’t think the ability to translate coding_seq is a high priority feature for this toolbox. However, the way it works now leads to inaccurate reporting. I know enough to generally ignore that quilt for every analysis, but I provide your reports to my users all the time, and I’m sure they’ll be confused as to why their cell line/mouse/therapeutic strategy is disrupting the protein sequence. Manipulating the the coding_seq is not an option, as it will then lead to inaccurate reporting of frame-shifting mutations, which IS a core feature of this toolbox, critical to the fundamental goal of CRISPResso analysis. I always assumed this is why this feature was not implemented sooner. A possible solution is to provide all 6 reading ORFS for each read, but that’s too unwieldy. My only request would be an option to suppress the quilt. (This would be the default in my wrapper). I’d love to hear your thoughts. Thanks! Dustin

…

On Apr 30, 2026, at 6:31 PM, Cole Lyman ***@***.***> wrote: Hi Dustin, Thanks for the explanation, for now we will support this by recommending that you set your coding_seq to be the start of the reading frame, and supplying the RC of the amplicon to support coding_seqs that may be antisense to the amplicon. The main motivation behind this decision is to try to keep complexity low (and not add yet another parameter to CRISPResso). Thank you for the feedback! Cole — Reply to this email directly, view it on GitHub <https://urldefense.com/v3/__https://github.com/pinellolab/CRISPResso2/discussions/642*discussioncomment-16774991__;Iw!!Mak6IKo!LkpvEZLf7ZYZ0Pi49-rB4DutBKgfSsHylsVAYdciRTD6-4Wj4ZEqJ-6FCw3Ufnr0LoKHt_o4WUOz6dc5KavLzJdpcik$>, or unsubscribe <https://urldefense.com/v3/__https://github.com/notifications/unsubscribe-auth/BHVZEP5IKCQK7U3JK7T36U34YPO6XAVCNFSM6AAAAACYGFDCAWVHI2DSMVQWIX3LMV43URDJONRXK43TNFXW4Q3PNVWWK3TUHMYTMNZXGQ4TSMI__;!!Mak6IKo!LkpvEZLf7ZYZ0Pi49-rB4DutBKgfSsHylsVAYdciRTD6-4Wj4ZEqJ-6FCw3Ufnr0LoKHt_o4WUOz6dc5KavLXur7HUE$>. Triage notifications on the go with GitHub Mobile for iOS <https://urldefense.com/v3/__https://apps.apple.com/app/apple-store/id1477376905?ct=notification-email&mt=8&pt=524675__;!!Mak6IKo!LkpvEZLf7ZYZ0Pi49-rB4DutBKgfSsHylsVAYdciRTD6-4Wj4ZEqJ-6FCw3Ufnr0LoKHt_o4WUOz6dc5KavLW1efl1o$> or Android <https://urldefense.com/v3/__https://play.google.com/store/apps/details?id=com.github.android&referrer=utm_campaign*3Dnotification-email*26utm_medium*3Demail*26utm_source*3Dgithub__;JSUlJSU!!Mak6IKo!LkpvEZLf7ZYZ0Pi49-rB4DutBKgfSsHylsVAYdciRTD6-4Wj4ZEqJ-6FCw3Ufnr0LoKHt_o4WUOz6dc5KavLX1PLkLo$>. You are receiving this because you authored the thread.