# SARS-CoV-2 E484K mutation and del 69-70 deletion **URL:** \ **Category:** General\ **Created:** [May 5, 2023, 6:36pm UTC](https://nextstrain.discourse.group/t/sars-cov-2-e484k-mutation-and-del-69-70-deletion/1378 "2023-05-05T18:36:10Z")\ **Posts on this page:** 8\ **Page:** 1
**Author:** ![lucyintheskyzzz](https://avatars.discourse-cdn.com/v4/letter/l/74df32/32.png) [@lucyintheskyzzz](https://nextstrain.discourse.group/u/lucyintheskyzzz)\ **Post date:** [May 5, 2023, 6:36pm UTC](https://nextstrain.discourse.group/t/sars-cov-2-e484k-mutation-and-del-69-70-deletion/1378/1 "2023-05-05T18:36:10Z")
Hi, I am using IDSeq/Nextclade ![Screenshot 2023-05-05 133228](https://canada1.discourse-cdn.com/flex031/uploads/nextstrain/original/1X/f86b52f3a8572900e1533dd66e53bec08264ff56.png) to identify my SARS-CoV-2 variants and export all the S-protein mutation regions to see what was covered in my reads. We noticed that the E484K and deletion 69-70 regions were not reported in the output. Here is an output for mostly B.1.1.7 and the deletion 69-70 region was not reported. Does Nextclade report these regions? ---
**Author:** ![corneliusroemer](https://yyz1.discourse-cdn.com/flex031/user_avatar/nextstrain.discourse.group/corneliusroemer/32/175_2.png) [@corneliusroemer](https://nextstrain.discourse.group/u/corneliusroemer)\ **Post date:** [May 5, 2023, 8:47pm UTC](https://nextstrain.discourse.group/t/sars-cov-2-e484k-mutation-and-del-69-70-deletion/1378/2 "2023-05-05T20:47:39Z")
Hi Lucy! It’s hard to say definitively why S:E484K is not shown. The mutation is either not present in your sequence, or that region is unsequenced (N). Can you share the sequence name/accession number with me so I can have a look? You can see whether the region is sequenced or not by switching to “Gene S” view using the gene view dropdown: ![image](https://canada1.discourse-cdn.com/flex031/uploads/nextstrain/original/1X/f95a10f4d4cc32e7603aebf7d328a9f6086c6b8a.jpeg) If it’s greyed out, then a region is unsequenced, in my case S:229-289 is unsequenced. ![image](https://canada1.discourse-cdn.com/flex031/uploads/nextstrain/original/1X/39be1453ee74799e490f01e32a97d4947bc55d68.png) You can check what regions are Ns by hovering over the number in the “Ns” column, then you get a tooltip that will show which regions are Ns: ![image](https://canada1.discourse-cdn.com/flex031/uploads/nextstrain/original/1X/5968b6baeec296ea9bd2f4786770690b1b4d2f30.png) Deletions are shown in the column called “Gaps”: ![image](https://canada1.discourse-cdn.com/flex031/uploads/nextstrain/original/1X/abc4b80d25c8d833fe5bc1eed6fea4f578b3bb64.png) I hope that makes things clearer. Let me know if you have any questions. If you have any particular questions regarding IDSeq, you could try asking here in their repo: [GitHub - chanzuckerberg/czid-web: Infectious Disease Sequencing Platform](https://github.com/chanzuckerberg/czid-web), I’m not familiar with IDSeq, only with Nextclade. Best, Cornelius ---
**Author:** ![lucyintheskyzzz](https://avatars.discourse-cdn.com/v4/letter/l/74df32/32.png) [@lucyintheskyzzz](https://nextstrain.discourse.group/u/lucyintheskyzzz)\ **Post date:** [July 2, 2023, 10:01pm UTC](https://nextstrain.discourse.group/t/sars-cov-2-e484k-mutation-and-del-69-70-deletion/1378/3 "2023-07-02T22:01:16Z")
Hi Cornelius, I ran all my sequences in IDSeq which in turn uses Nextclade, how can I find the accession number? Katie ---
**Author:** ![lucyintheskyzzz](https://avatars.discourse-cdn.com/v4/letter/l/74df32/32.png) [@lucyintheskyzzz](https://nextstrain.discourse.group/u/lucyintheskyzzz)\ **Post date:** [July 2, 2023, 10:03pm UTC](https://nextstrain.discourse.group/t/sars-cov-2-e484k-mutation-and-del-69-70-deletion/1378/4 "2023-07-02T22:03:51Z")
Hi Cornelius, I was also wondering what are the recommended % genome recoveries necessary for accurate SARS-CoV-2 variant calling using NextClade? I was thinking you could get a low % genome recovery from the consensus alignment, but maybe your primers sequenced key regions for accurate variant calling. What key regions on the SARS-CoV-2 genome should be sequenced in order for NextClade to accurately call a variant? Thanks! ---
**Author:** ![corneliusroemer](https://yyz1.discourse-cdn.com/flex031/user_avatar/nextstrain.discourse.group/corneliusroemer/32/175_2.png) [@corneliusroemer](https://nextstrain.discourse.group/u/corneliusroemer)\ **Post date:** [July 14, 2023, 11:31am UTC](https://nextstrain.discourse.group/t/sars-cov-2-e484k-mutation-and-del-69-70-deletion/1378/5 "2023-07-14T11:31:57Z")
> I ran all my sequences in IDSeq which in turn uses Nextclade, how can I find the accession number? I’m not quite sure what you mean by accession number, as Nextclade just looks at the sequences you give it. I don’t know how IDSeq uses Nextclade. Maybe you could ask at an IDSeq forum? Or show me a screenshot and explain in more detail what you mean. Happy to help! I’m actually curious how IDSeq incorporates Nextclade, so if you could share a bit that would be super interesting. ---
**Author:** ![corneliusroemer](https://yyz1.discourse-cdn.com/flex031/user_avatar/nextstrain.discourse.group/corneliusroemer/32/175_2.png) [@corneliusroemer](https://nextstrain.discourse.group/u/corneliusroemer)\ **Post date:** [July 14, 2023, 11:33am UTC](https://nextstrain.discourse.group/t/sars-cov-2-e484k-mutation-and-del-69-70-deletion/1378/6 "2023-07-14T11:33:16Z")
I answered here: > [@NextClade Variant Calling info](https://nextstrain.discourse.group/t/nextclade-variant-calling-info/1414/2): > > Hi Katie! That’s a great question. There is no single answer for what proportion of the genome allows accurate variant calling. In general, the higher the coverage, the more confident/accurate the answer. It all depends on what you’re trying to achieve. If you give some more details about what you’re planning to do I will happily share some tailored advice. In general, as most of the important antigenic evolution happens in Spike, it’s very useful to cover Spike, in particular everything betw… Why do you get low % genome recovery? Are you working with waste water and trying to generate a consensus from a waste water sequence? Or are you trying to be more cost-effective and only sequence parts of the genome of patient samples? ---
**Author:** ![lucyintheskyzzz](https://avatars.discourse-cdn.com/v4/letter/l/74df32/32.png) [@lucyintheskyzzz](https://nextstrain.discourse.group/u/lucyintheskyzzz)\ **Post date:** [July 18, 2023, 9:23pm UTC](https://nextstrain.discourse.group/t/sars-cov-2-e484k-mutation-and-del-69-70-deletion/1378/7 "2023-07-18T21:23:34Z")
Hi @corneliusroemer here is the link to the IDSeq pipelines for SARS-CoV-2 [CZ ID Pipeline Overviews – CZ ID Help Center (zendesk.com)](https://chanzuckerberg.zendesk.com/hc/en-us/articles/13756558532884-CZ-ID-Pipeline-Overview#mNGS-Nanopore-overview) [View SARS-CoV-2 Genomes in Nextclade – CZ ID Help Center (zendesk.com)](https://chanzuckerberg.zendesk.com/hc/en-us/articles/360052479232-View-SARS-CoV-2-Genomes-in-Nextclade) ---
**Author:** ![lucyintheskyzzz](https://avatars.discourse-cdn.com/v4/letter/l/74df32/32.png) [@lucyintheskyzzz](https://nextstrain.discourse.group/u/lucyintheskyzzz)\ **Post date:** [July 18, 2023, 9:38pm UTC](https://nextstrain.discourse.group/t/sars-cov-2-e484k-mutation-and-del-69-70-deletion/1378/8 "2023-07-18T21:38:56Z")
Hi @corneliusroemer thanks so much for your response this is so helpful! I am working on a paper, and we basically sequencing wastewater for SARS-CoV-2 using minION mk1b using the ARTIC v3 and v4.1 primers. We also used RT-ddPCR with the GT Molecular SARS-CoV-2 kit for variant calling. We analyzed all our data and noticed that we get 50%-75% genome recovery when the N1 and N2 genes are \>30k GC/100mL and 75%-100% when \>48k GC/100mL. I guess I just wanted to know what the best % genome recovery gives you the most accurate variant calling (for the discussion part of the paper). What is a good cutoff % recovery? I did a rapid literature review search and most publications are setting their % genome recovery to \>90%. I just wanted to know your input, since you are an expert on how NextClade works. Katie Thanks! Katie