# H7 hemmaglutinin phylogeny showing FCS
**URL:** https://nextstrain.discourse.group/t/h7-hemmaglutinin-phylogeny-showing-fcs/527
**Category:** General
**Created:** [May 10, 2021, 12:29pm UTC](https://nextstrain.discourse.group/t/h7-hemmaglutinin-phylogeny-showing-fcs/527 "2021-05-10T12:29:31Z")
**Posts on this page:** 5
**Page:** 1
### Author:  [@babarlelephant](https://nextstrain.discourse.group/u/babarlelephant)
#### Post date: [May 10, 2021, 12:29pm UTC](https://nextstrain.discourse.group/t/h7-hemmaglutinin-phylogeny-showing-fcs/527/1 "2021-05-10T12:29:31Z")
Hi, Influenza H7 hemmaglutinin phylogenies are a bit complicated due to many indels at the most interesting portion: the furin cleavage site which is a key determinant of low/high pathogenicity.
My current methodology is:
- to blast on ncbi a full-length H7/HA sequence and obtain the fasta of the 1720 sequences with very good cover,
- to take as reference CY130150 having a large FCS and to call augur align without fill-gaps,
- to edit the alignement with aliview and notepad++ moving several nucleotides to the left or right of the deletions to make the deletions in-frame,
- to run MEGA/minium evolution tree to obtain a raw newick tree,
- to call augur refine, augur ancestral --keep-ambiguous, augur translate, augur export
You can see the result there [http://babarlelephant.free-hoster.net/dist/index\_H7.html?c=gt-HA\_337,338,339,340,341](http://babarlelephant.free-hoster.net/dist/index_H7.html?c=gt-HA_337,338,339,340,341)
The whole process is a bit hard to reproduce. My main concerns:
- What is your solution for making the deletions in-frame?
- Several sequences have an insertion, often not exactly at the FCS, making the FCS deletion not a multiple of 3, as the insertion is discarded in the alignement this is making the AA translation a bit sloppy for those sequences (those with a X in the [translation](http://babarlelephant.free-hoster.net/dist/index_H7.html?c=gt-HA_337,338,339,340,341))
- Why is IQTREE giving a poor result in contrary to MEGA ?
It is not a problem with the alignement which is very good. I didn’t try yet to do a fill-gap only for the missing nucleotides at the beginning and end of the sequences.
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### Author:  [@babarlelephant](https://nextstrain.discourse.group/u/babarlelephant)
#### Post date: [May 10, 2021, 2:35pm UTC](https://nextstrain.discourse.group/t/h7-hemmaglutinin-phylogeny-showing-fcs/527/2 "2021-05-10T14:35:04Z")
For IQTREE it might mainly a rooting problem.
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### Author:  [@babarlelephant](https://nextstrain.discourse.group/u/babarlelephant)
#### Post date: [May 12, 2021, 2:02pm UTC](https://nextstrain.discourse.group/t/h7-hemmaglutinin-phylogeny-showing-fcs/527/3 "2021-05-12T14:02:06Z")
There is a tree for H5 [http://babarlelephant.free-hoster.net/dist/index\_H5.html?c=gt-HA\_341,342,343,344,345,346,347](http://babarlelephant.free-hoster.net/dist/index_H5.html?c=gt-HA_341,342,343,344,345,346,347)
This time I downloaded the data from [http://openflu.vital-it.ch/](http://openflu.vital-it.ch/) (all the full length H5 sequences).
MEGA gave a not too bad result except for one H5N6 clade, and I am making the deletions in-frame with a simple script. For the alignement I am calling mafft --keeplength --addfragments sequences.fasta reference.fasta \> aligned.fasta
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### Author:  [@Vetdog](https://nextstrain.discourse.group/u/Vetdog)
#### Post date: [November 12, 2021, 2:32pm UTC](https://nextstrain.discourse.group/t/h7-hemmaglutinin-phylogeny-showing-fcs/527/4 "2021-11-12T14:32:35Z")
For align using MAFFT, `mafft --genafpair --maxiterate 1000 input.fas > output.fas` may also give you a good alignment result. If the sequence quality was satisfied, using mafft (within Phylosuite or Bioaider) under codon alignment model may also give you an in-frame result.
Otherwise, using augur mask the inferior alignment sites.
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### Author:  [@rneher](https://nextstrain.discourse.group/u/rneher)
#### Post date: [November 13, 2021, 9:06am UTC](https://nextstrain.discourse.group/t/h7-hemmaglutinin-phylogeny-showing-fcs/527/5 "2021-11-13T09:06:52Z")
The translation problems with indels are one of the primary reasons we developed nextalign. this would try to place indels such that they align with codons and produce a separate amino acid alignment that can be used to annotate the tree with amino acid sequences and mutations. It will still strip insertions though.