Multiple sequence alignments

Sunday 31 October 2021

Multiple sequence alignments

A sequence alignment is a rather important tool.
  • Sequence conservation is a key ingredient in most nucleotide mutation severity predictors.
  • The covariance within it powers the AlphaFold2 Evoformer and other de novo structure predictors.
  • The phylogeny extracted from it tells the evolutionary tale of the protein
However, on the very basic level, i.e. getting a nice figure, far from the world of covariance matrices, it is a slight nuisance.
Therefore I would like share some pointers on choosing species and two python operation, namely getting the equivalent residue in a homologue and making a figure in Plotly. Just like with docking, where careful and diligent human choices make all the difference, rational choices help greatly with clarity for sequence alignments.


This post isn't aimed at going through all the basics of making a MSA, but rather focus on three details mentioned and in particular is focused on animals and not other groups. But briefly, to make MSA, Muscle (web, binary) is very robust. if there are too many sequences, Clustal Omega is a good choice and cd-hit at removing diversity —akin to using a uniref70, 50 or 30. For tree inference, there are many different options, using different approaches whose discussion has filled textbooks. But RAxML is really good, but if a quick neighbour joined tree is required, EBI has an web tool for it.

Footnote on lead

In the lead I mention nucleotide mutation severity predictors, e.g. CADD scores. These just spit out a mysterious number, whereas it is a lot more informative looking where the residue is in the structure —I may be biased as I wrote Venus, a tool to investigate exactly that. But generally buried core residues are conserved, whereas surface residues are less —unless they are interface residues, conformationally important or involved in some other mechanism!

In terms of mapping sequence conservation on structure, Consurf is the tool to use. However, even if I prefer the former, a MSA figure is the preferred option for sequence conservation in publications as it says explicitly, how far back is it conserved even though it does not show secondary structure and surface exposure.

Making the MSA itself: humans

A rather key ingredient in making the multiple sequence alignment (MSA) itself is choosing the species.
For high-throughput projects this is easy: blast and remove redundancy, but MSAs for human readers, it is not. Ideally this should be well spaced out and species that a reader can recognise.

For animal protein trees these near-universally follow the species tree, with the exception of gene duplication events.
As a result the assembly of the group of sequences requires going back along the evolutionary tree and picking species whose ancestor branched off earlier and earlier —but with a strong focus on genome duplication events as these may be when the gene was duplicated.
Doing a BLAST search will return an endless troop of monkeys and conspiracy of lemurs, which lacks divergence, so often a manual selection is more useful for figures. This has four points to note.

1. Genome quality

A big problem is that the genome of many phylogenetically useful animals (e.g. coral, sea urchin, wallaby, dog etc.) may not be as well curated as one would hope, resulting in shorter protein (missing exons). Mice (rodents) and rabbits (langomorphs) are a sister clade to monkeys and lemurs, so are not as distant as one would think, however mice are a model organism, therefore their genome is well curated, so are often seen included in MSAs albeit often pointlessly.
The annotated name is highly variable, therefore doing a Blast search filtered for the particular species of interest is the better approach.

2. NCBI or Uniprot?

I normally use Uniprot because the curation is better for model organisms, but NCBI is way better curated than Uniprot for non-model organism. In NCBI, there may be multiple entries with different lengths, these may be different isoforms or a paralogue —if from a blast search the identify percentage will be similar for the former, but not the latter.

3. Sidenote: common name or scientific name?

The scientific names in a Blast search:
  • Sus → suine = pig
  • Tichochus → walrus = tricheco in Italian
  • Vombatus → Wombat. Cute Scientific name!
  • Nyctibius → nyx is night so... bat? No. it's a bird

I personally prefer using the common name of the species or better the single word name of a parent taxon, because to me most scientific names are gibberish. I can speak Italian, which makes guessing some less common Latin names easier, for example a blackbird is a «merlo» and its Latin name is Turdus merula*, but most are still alien, for example a red kite is a «nibbio reale», but its Latin name is Milvus milvus.

*) In the above sentence, I should have said "Eurasian blackbird" if I wanted to be pedantically precise, but for most applications this is just pointless noise...

I am also of the opinion that using kelvin in an enzymology paper is utterly ridiculous, but I have been told this is a sign I am a rubbish scientist. So as said, preference for intelligible common names is my personal opinion and I may be a bad scientist for it!

4. Animal phylogeny

Choosing non-model animals requires a crash course on animal phylogeny and in particular when genome duplication events happened. There are some organisms that would be useful, but these are not genome sequenced, therefore some limitations are at play. Also, if there is no gene in one of them, it may be an annotation or coverage issue, so checking sister species is ideal if possible —the number of unsequenced animals is large: the adorable mexican axolotl is the sole sequenced salamander, while no newt is. 

Some watery organisms stand out:
  • Lamprey (Petromyzon marinus) are the thing of nightmares, but predate the genome double duplication event in jawed fish, so are very nice phylogenetically as often ends up being the outgroup. When a gene family is still duplicated in lampreys one can go back further:
  • Sea urchin (Strongylocentrotus purpuratus) is basal to vertebrates. It is an invertebrate, but not of insect, molluscs and worms kind (deuterostome not protostome). So it is more closely related to us than insects, molluscs and worms.
  • Coral (Acropora millepora) is an animal that is basal to all others.
  • Zebrafish (Danio rerio) is a ray finned fish with a curated genome. Most ray-finned fish however descent from an ancestor with a genome duplication event. Tetrapods (land vertebrates) evolved from lobe finned fish, for which the extant examples are lung fish and coelacanths. 
  • Coelacanth (Latimeria chalumnae) is not a Pokémon found in New Zealand —that's Relicanth—but a fish basal to tetrapods. Together ray-finned fish and lobe-finned fish form the boney fish, whose common ancestors had two genome duplication events, once after lampreys diverged and the second possibly after sharks (jawed cartilaginous fish).
  • Great white shark (Carcharodon carcharias) is a sequenced shark along with two others.
  • Sturgeon (Acipenser ruthenus) is a basal ray-finned fish that predates the genome duplication seen in zebrafish. For the purpose of diversity of a gene that duplicated in lampreys, fish are really useful as they are diverse despite looking all the same —so picking some fish near randomly  would work. Do note that zebrafish is a minnow, which is in the same family as carp (sequenced).
  • Axolotl (Ambystoma mexicanum) is an amphibian, which are basal tetrapods.

Tetrapod phylogeny after amphibians is straightforward as there are two clades one with reptiles and birds, the other with mammals. As a result, using one representative for the former, say chicken (Gallus gallus), would suffice. Parenthetically, I should say that all extant species have evolved to the present day, even if they look primitive, so there's no difference in terms of divergence distance from humans between an iguana and an eagle.
In mammals, the phylogeny is well known. The basal monotremes platypus (Ornithorhynchus anatinus) and echidna (not sequenced) lay eggs and marsupials, such as a wallabies (Notamacropus eugenii, no big kangaroos sequenced) and wombats (Vombatus ursinus) have pouches. Then there's a radiation of different placental mammals, which means that pinning the precise relationship between the clades has been tricky and that the representative sequence divergence is not going to suffer if the chosen representative species is not quite basal—for a detailed review of mammalian phylogeny see this review. Also, if someone is interested in showing a protein MSA, the best example of sequence conservation/divergence is further down the tree. Two things need noting, mammalian carnivores form a single clade and many mammals that come to mind are from this clade, while cloven animals and cetaceans form a clade (Cetartiodactyla)—this is not important, but I really wanted to mention that hippos and whales had a common ancestor called a "whippo" (hence the clade name Whippomorpha), which is a fantastic name.

Bacterial MSA

For bacteria, things get messy due to widespread horizontal gene transfer. So gene trees rarely match species trees. In their favour is the fact that their genomes are far easier to annotate and smaller, so there's a much larger diversity to choose from. However, they are much more diverse. As a result I cannot give pointers...

Python and MSAs

R has market dominance over Python when it comes to genetic bioinformatics, but generally the operations are simple, so there's not too much issue. In terms of most complete MSA plotting tool, that would be Textshade in Latex —I dislike it as it is highly complicated, I seem to want to some near-impossible corner case which if done from scratch may have been easier and I am addicted to Jupyter notebooks...

Starting from the basics, retrieving sequences from NCBI is easy with BioPython:

from Bio import Entrez = "👻@👻.👻"
Entrez.api_key = "👾👾👾👾"  # you get this by registering
with Entrez.efetch(db="protein", id=target, rettype="fasta", retmode="text") as handle:
    fasta =

Likewise, with UniProt it is even easier:

url = f'{uniprot_id}.fasta'
reply = requests.get(url)
assert reply.status_code == 200, f'{reply.status_code}: {url}'
fasta = reply.text

One thing to note is that Uniprot and NCBI have different header format, for example here are two functions to get information from them (without using BioPython):

def split_ncbi_fasta(fasta:str) -> dict: # keys: ['accession', 'description', 'species', 'sequence']
  return re.match('\&gt;(?P<accession>\S+)\s+(?P<description>.*)\s+\[(?P<species>.*)\](?P<sequence>\w*)', fasta.replace('\n','')).groupdict()
def split_uniprot_fasta(fasta:str) -> dict: # keys: ['type', 'accession', 'name', 'description', 'args', 'OS' = species, 'OX', 'GN', 'PE', 'sequence']
match = re.match(r'\>(?P<type>\w+)\|(?P<accession>.*?)\|(?P<name>[\w\_]+)\s(?P<description>.*?)\s(?P<args>\w+\=.*)', fasta).groupdict() details = dict(re.findall(r'(\w+)\=(.*?)\s+\n', # there is a way to split this more cleanly... re.sub(r'(\w+\=)', r'\n\1', match['args']+'\n') ) ) return {**match, **details, 'sequence': ''.join(fasta.split('\n')[1:])}

The next few operations (i.e. writing a fasta file with headers of one's choice, aligning locally via a muscle3 system call or remotely via Muscle server or any other aligner and reading the aligned sequences again) are very straightforward, but have a few personal choices, e.g. how to clean the labels, whether to use pandas or dictionaries etc.

One operation that is not too straightforward is getting the equivalent residue position between two sequences which requires one to remember to correct the position as they aren't zero-indexed making it an otherwise simple operation namely creating a list where each index corresponds to the zero-indexed ungapped position of the sequence, while the elements are the index of these in the alignment:

def get_mapping(seq: str) -> List[int]:
    """Given a sequences with gaps return a zero-index
    list mapping ungapped position --> gapped position """
    return [i for i, p in enumerate(seq) if p != '-']

def convert(query_seq: str, target_seq: str, position: int) -> int:
    Given a query sequence (aligned) and target sequence (aligned)
    and a ungapped query position, return the ungapped target position
    :param query_seq: aligned
    :param target_seq: aligned
    :param position: query residue number counted from 1
    :return: target residue number counted from 1
    query_mapping = get_mapping(query_seq)
    target_mapping = get_mapping(target_seq)
    mp = query_mapping[position - 1]
    return target_mapping.index(mp) + 1

The next thing is the figure. This is actually really straightforward and can be done with a scatterplot, where the datapoints are a residue represented by label without the marker shown and the position is to make a grid. For example:

import plotly.graph_objects as go
import numpy as np
from collections import Counter
from typing import *

def make_fig(al_seqs: Dict[str, str], position: int, names: Optional[List[str]] = None) -> go.Figure:
    This make a fake scatter plot with letters for each position in Plotly.

    :param al_seqs: aligned seq dictionary of names to seqs w/ gaps (aligned)
    :param position: position to show based on the first name
    :param names: order or the seqs to show top to bottom —if subset only these will be shown
    # --------------------------------------------------
    # prep
    if names is None:
        names = list(al_seqs.keys())
    elif len(names) != len(al_seqs):
        sub_al_seqs = {name: [] for name in names}
        for i in range(len(al_seqs[names[0]])):
            if any([al_seqs[name][i] != '-' for name in names]):
                for name in names:
        for species in sub_al_seqs:
            sub_al_seqs[species] = ''.join(sub_al_seqs[species])
        al_seqs = sub_al_seqs
    # mapping
    first = al_seqs[names[0]]
    n = len(first)
    mapping = get_mapping(first)
    position = position - 1
    start = mapping[position] - 10
    mid = mapping[position]
    stop = mapping[position] + 10
    # out of bonds prevention
if start < 0: forepadding = ['>'] * abs(start) mid += abs(start) start = 0 else: forepadding = [] if stop > n: aftpadding = ['<'] * stop - n stop = n else: aftpadding = [] # -------------------------------------------------- # Add letters scatters = [go.Scatter(x=np.arange(start, stop + 1), # y=[-i/yzoom] * 20, y=[name] * 21, text=forepadding + list(al_seqs[name][start:stop + 1]) + aftpadding, textposition="middle center", # textfont_size=10, textfont=dict( family="monospace", size=12, color="black" ), name=name, mode='text') for i, name in enumerate(names) ] # -------------------------------------------------- # Add red box fig = go.Figure(data=scatters) fig.add_shape(type="rect", x0=mid - 0.5, x1=mid + 0.5, # y0=0.5/yzoom, y1=(0.5 -len(al_seqs))/yzoom, y0=0 - 0.5, y1=len(names) - 1 + 0.5, # the len is off-by-one line=dict(color="crimson"), ) fig.update_shapes(dict(xref='x', yref='y')) # -------------------------------------------------- # Add turquoise squares for i in range(start, stop + 1): residues = [al_seqs[name][i] for name in names] mc = Counter(residues).most_common()[0][0] for j, r in enumerate(residues): if mc == '-': continue elif r == mc: fig.add_shape(type="rect", x0=i - 0.5, x1=i + 0.5, # y0=(-j -.5)/yzoom, y1=(-j +.5)/yzoom, # y0=names[j], y1=names[j], y0=j - 0.5, y1=j + 0.5, fillcolor="aquamarine", opacity=0.5, layer="below", line_width=0, ) # -------------------------------------------------- # Correct fig.update_layout(template='none', showlegend=False, title=f'Residue {position + 1}', xaxis={ 'showgrid': False, 'zeroline': False, 'visible': False, # 'range': [start,stop], }, yaxis={ 'showgrid': False, 'zeroline': False, 'visible': True, 'autorange': 'reversed' } ) # -------------------------------------------------- return fig

This would make something like:

By doing it this way one can easily change how the plot is made, such as colour only residues that match the first one or make the labels slanted (fig.update_layout(yaxis=dict(tickangle = -45) )). One thing to note however is that the above function makes only a window and if the whole sequences are used and zoomed in (via the range argument to the layout of xaxis) one can zoom and scroll around, but the figure becomes very heavy due to the added shapes (turquoise background).

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