# Nextalign | Error: Invalid nucleotide: "L"

**URL:** <https://nextstrain.discourse.group/t/nextalign-error-invalid-nucleotide-l/535>\
**Category:** Uncategorized\
**Created:** [May 18, 2021, 12:46pm UTC](https://nextstrain.discourse.group/t/nextalign-error-invalid-nucleotide-l/535 "2021-05-18T12:46:58Z")\
**Posts on this page:** 2\
**Page:** 1

<div class="post-metadata">

**Author:** ![andersonbrito](https://yyz1.discourse-cdn.com/flex031/user_avatar/nextstrain.discourse.group/andersonbrito/32/72_2.png) [@andersonbrito](https://nextstrain.discourse.group/u/andersonbrito)\
**Post date:** [May 18, 2021, 12:46pm UTC](https://nextstrain.discourse.group/t/nextalign-error-invalid-nucleotide-l/535/1 "2021-05-18T12:46:58Z")

</div>

Hi,

I’m running the latest version of `nextalign`, available [here](https://github.com/nextstrain/nextclade/tree/master/packages/nextalign_cli). Below you can find the command I’m running:

> nextalign-MacOS-x86\_64 --sequences sequences\_test.fasta --reference reference.gb --genemap genemap.gff --genes E,M,N,ORF10,ORF14,ORF1a,ORF1b,ORF3a,ORF6,ORF7a,ORF7b,ORF8,ORF9b,S --output-dir output\_files/ --output-basename test

I’m getting an `Error: Invalid nucleotide: "L"` that claims one of my sequences have an `L` somewhere, but in fact no letter `L` (neither upper of lowercase) is found in the file, not even in sequence headers. Any idea what may be happening?

I’m getting this error using a multifasta with these 10 genomes:

> Wuhan/Hu-1/2019  
> HongKong/HKPU19\_0402/2020  
> USA/WI-UW-159/2020  
> USA/WI-UW-237/2020  
> Wuhan/WH01/2019  
> Ghana/2828\_S6/2020  
> France/GES-2843/2020  
> Singapore/95/2020  
> USA/CA-CZB-1088/2020  
> USA/IA-GMF-M00005/2020

Thank you

---

<div class="post-metadata">

**Author:** ![james](https://yyz1.discourse-cdn.com/flex031/user_avatar/nextstrain.discourse.group/james/32/11_2.png) [@james](https://nextstrain.discourse.group/u/james)\
**Post date:** [May 18, 2021, 11:03pm UTC](https://nextstrain.discourse.group/t/nextalign-error-invalid-nucleotide-l/535/2 "2021-05-18T23:03:27Z")

</div>

Solved by @rneher – the reference sequence must be FASTA. [Here’s](https://github.com/nextstrain/ncov/blob/master/defaults/reference_seq.fasta) the nCoV reference we are using.