# Using --existing-alignment During Augur Align **URL:** \ **Category:** Help and Getting Started\ **Created:** [July 22, 2020, 9:56pm UTC](https://nextstrain.discourse.group/t/using-existing-alignment-during-augur-align/81 "2020-07-22T21:56:57Z")\ **Posts on this page:** 9\ **Page:** 1
**Author:** ![colejensen](https://avatars.discourse-cdn.com/v4/letter/c/49beb7/32.png) [@colejensen](https://nextstrain.discourse.group/u/colejensen)\ **Post date:** [July 22, 2020, 9:56pm UTC](https://nextstrain.discourse.group/t/using-existing-alignment-during-augur-align/81/1 "2020-07-22T21:56:58Z")
Hello! I’ve been trying to use the --existing-alignment subcommand in augur align. With smaller previous alignments the command works and the build run smoothly (less than 100 total genomes), but when I use larger previous alignments I get the following error: ERROR: b’’ shell exited 1 when running: mafft --add results/aligned.fasta.to\_align.fasta --keeplength --reorder --anysymbol --nomemsave --adjustdirection --thread 1 config/aligned.fasta.ref.fasta 1\> results/aligned.fasta 2\> results/aligned.fasta.log Error during alignment [Wed Jul 22 17:36:11 2020] Error in rule align: jobid: 10 output: results/aligned.fasta shell: ``` augur align --existing-alignment config/aligned.fasta --sequences results/filtered.fasta --reference-sequence config/reference.gb --output results/aligned.fasta --remove-reference --fill-gaps (one of the commands exited with non-zero exit code; note that snakemake uses bash strict mode!) ``` Removing output files of failed job align since they might be corrupted: results/aligned.fasta Shutting down, this might take some time. Exiting because a job execution failed. Look above for error message. It’s worth noting that the build will run without error if I remove the ‘–existing-alignment’ line in the snakefile. Has anyone run into this issue before? ---
**Author:** ![trs](https://yyz1.discourse-cdn.com/flex031/user_avatar/nextstrain.discourse.group/trs/32/5_2.png) [@trs](https://nextstrain.discourse.group/u/trs)\ **Post date:** [July 22, 2020, 10:59pm UTC](https://nextstrain.discourse.group/t/using-existing-alignment-during-augur-align/81/2 "2020-07-22T22:59:24Z")
Hi @colejensen, welcome. 🙂 I expect there’s more detail about the error in the log file `results/aligned.fasta.log`. Can you share that with us? ---
**Author:** ![colejensen](https://avatars.discourse-cdn.com/v4/letter/c/49beb7/32.png) [@colejensen](https://nextstrain.discourse.group/u/colejensen)\ **Post date:** [July 23, 2020, 2:18am UTC](https://nextstrain.discourse.group/t/using-existing-alignment-during-augur-align/81/3 "2020-07-23T02:18:47Z")
Hi @trs. This is the entirety of the results/aligned.fasta.log: “Check results/aligned.fasta.to\_align.fasta”. The aligned.fasta.to\_align.fasta file is empty. ---
**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:** [July 25, 2020, 2:44am UTC](https://nextstrain.discourse.group/t/using-existing-alignment-during-augur-align/81/4 "2020-07-25T02:44:05Z")
Hey, Last time I saw that error, it was caused by a corrupted fasta file with missing new line characters, which merged two genomes, like shown below. > GGGCTATATAAACGTTTTCGCTTTTCCGTTTACGATATATAGTCTACTCTTGTGCAGAATGAATTCTCGTAACTACATAGCACAAGTAGATGTAGTTAACTTTAATCTCACATAGCAATCTTTAATCAGTGTGTAACATTAGGGAGGACTTGAAAGAGCCACCACATTTTCACCGAGGCCACGCGGAGTACGATCGAGTGTACAGTGAACAATGCTAGGGAGAGCTGCCTATATGGAAGAGCCCTAATGTGTAAAATTAATTTTAGTAGT\>HCOV-19/FINLAND/5MAY46S12/2020|EPI\_ISL\_481700|2020-05-05NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAGATCTGTTCTCTAAACGAACTTTAAAATCTGTGTGGCTGTCACTCGGCTGCATGCTTAGTGCACTCACGCAGTATAATTAATAACTAATTACTGTCGTTGACAGGACACGAGTAACTCGTCTATCTTCTGCAGGCTGCTTACGGTTTCGTCCGTGTTGCAGCCGATCATCAGCACATCTAGGTTTT ---
**Author:** ![colejensen](https://avatars.discourse-cdn.com/v4/letter/c/49beb7/32.png) [@colejensen](https://nextstrain.discourse.group/u/colejensen)\ **Post date:** [July 27, 2020, 5:36pm UTC](https://nextstrain.discourse.group/t/using-existing-alignment-during-augur-align/81/5 "2020-07-27T17:36:19Z")
Hi @andersonbrito, I can confirm that the alignment file used that produced this error doesn’t have merged genomes. ---
**Author:** ![trs](https://yyz1.discourse-cdn.com/flex031/user_avatar/nextstrain.discourse.group/trs/32/5_2.png) [@trs](https://nextstrain.discourse.group/u/trs)\ **Post date:** [July 28, 2020, 5:09pm UTC](https://nextstrain.discourse.group/t/using-existing-alignment-during-augur-align/81/6 "2020-07-28T17:09:19Z")
> [@colejensen](#): > > The aligned.fasta.to\_align.fasta file is empty. Ah, I’d bet this is what mafft errors about. The question then is: 1. Are there really no new sequences to align? That is, `results/filtered.fasta` contains no sequences which aren’t in `config/aligned.fasta`? 2. or, does Augur have a bug which makes it incorrectly write out an empty “to align” file? Can you check on this? Or, if you provide the original input files to your `augur align` invocation, we can check. 🙂 ---
**Author:** ![colejensen](https://avatars.discourse-cdn.com/v4/letter/c/49beb7/32.png) [@colejensen](https://nextstrain.discourse.group/u/colejensen)\ **Post date:** [July 29, 2020, 2:48am UTC](https://nextstrain.discourse.group/t/using-existing-alignment-during-augur-align/81/7 "2020-07-29T02:48:58Z")
Here are the links to the inputs you asked for. > **[aligned.fasta](https://drive.google.com/file/d/1fJ3Eo3JSomLCCXjCoeQrS6t03dTzopSs/view?usp=sharing)** > > Google Drive file. > **[filtered.fasta](https://drive.google.com/file/d/1dHDqDfxrvaeNtPU5ULRU5XLYawa6QuNt/view?usp=sharing)** > > Google Drive file. > **[reference.gb](https://drive.google.com/file/d/1v55RByU6Yn2a8a6O2Sy__k_wYpLGwAnS/view?usp=sharing)** > > Google Drive file. There should be new sequences to align, but I’d love a second opinion. @trs thank you. ---
**Author:** ![trs](https://yyz1.discourse-cdn.com/flex031/user_avatar/nextstrain.discourse.group/trs/32/5_2.png) [@trs](https://nextstrain.discourse.group/u/trs)\ **Post date:** [July 29, 2020, 6:49pm UTC](https://nextstrain.discourse.group/t/using-existing-alignment-during-augur-align/81/8 "2020-07-29T18:49:53Z")
Hmm, there are definitely lots of new sequences to align in those files. The files also work fine for me when I run the following locally: ```bash augur align \ --existing-alignment ~/Downloads/aligned.fasta \ --sequences ~/Downloads/filtered.fasta \ --reference-sequence ~/Downloads/reference.gb \ --output /tmp/output.fasta \ --remove-reference \ --fill-gaps ``` I don’t know if it makes a difference, but what version of Augur are you using? `augur --version` will report it if you’re running Augur inside Conda. If you’re using the Nextstrain CLI, `nextstrain version --verbose` will report it. So far the only way I can reproduce the error you’re seeing and Mafft’s “Check results/aligned.fasta.to\_align.fasta” message is by setting up inputs such that there’s nothing new to align. ---
**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:** [October 10, 2020, 8:17pm UTC](https://nextstrain.discourse.group/t/using-existing-alignment-during-augur-align/81/9 "2020-10-10T20:17:34Z")
Hey, I’m not sure if you found a solution for this issue, but I often get that error when the sequences in `aligned.fasta` match exactly the content in `filtered.fasta`. The solution: try removing one of the sequences in `aligned.fasta`. MAFFT will only realign that extra sequence, and move on. ``` ERROR: b'' shell exited 1 when running: mafft --add results/aligned.fasta.to_align.fasta --keeplength --reorder --anysymbol --nomemsave --adjustdirection --thread 1 config/aligned.fasta.ref.fasta 1> results/aligned.fasta 2> results/aligned.fasta.log Error during alignment ``` It could have other causes, though. Cheers