Kille et al.

Genome Biology (2022) 23:182 Genome Biology

https://doi.org/10.1186/s13059-022-02735-6

REVIEW Open Access

Multiple genome alignment in the

telomere-to-telomere assembly era
Bryce Kille1 , Advait Balaji1 , Fritz J. Sedlazeck2 , Michael Nute1 and Todd J. Treangen1*

*Correspondence: [email protected]
1
Department of Computer Science, Abstract
Rice University, Houston, TX, USA
2
Human Genome Sequencing
With the arrival of telomere-to-telomere (T2T) assemblies of the human genome
Center, Baylor College of Medicine, comes the computational challenge of efficiently and accurately constructing multiple
Houston, TX, USA genome alignments at an unprecedented scale. By identifying nucleotides across
genomes which share a common ancestor, multiple genome alignments commonly
serve as the bedrock for comparative genomics studies. In this review, we provide an
overview of the algorithmic template that most multiple genome alignment methods
follow. We also discuss prospective areas of improvement of multiple genome
alignment for keeping up with continuously arriving high-quality T2T assembled
genomes and for unlocking clinically-relevant insights.
Keywords: Multiple genome alignment, Comparative genomics, Homology, Synteny

Background
The advent of new sequencing technologies has made available hundreds of high reso-
lution vertebrate genome assemblies and along with them the possibility of answering a
number of biological questions [1]. As a shining example of the potential of advances in
sequencing technologies coupled together with computational methods, in 2022 the com-
plete sequence of a human genome was published [2]. While a single genome alone can
yield numerous discoveries, it is the relationships among genomes which hold the key to
understanding genomic function and the evolutionary history behind it. These relation-
ships may be small variations, intergenomic distances, ultra-conserved or hyper-variable
regions, or structural variation, to name a few [3–7]. Multiple genome alignments are able
to accurately represent all of these relationships and more, serving as a rich platform for
numerous downstream avenues of comparative genomic analysis.
A useful starting point for discussing multiple genome alignment (MGA) is to first
define multiple sequence alignment (MSA). MSA is the method of assigning homol-
ogy relationships to nucleotides across 3 or more sequences (for 2 sequences, we will
use “pairwise” instead of “multiple”), where a set of nucleotides are homologous if they
descend from the same common ancestor. These alignments are typically represented by

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Kille et al. Genome Biology (2022) 23:182 Page 2 of 22

a 2-dimensional array where each row represents an input sequence and each column
represents a group of homologous nucleotides. MSA has the strict constraint that the
alignment is colinear, i.e., each row read from left to right, ignoring the empty columns,
must be the original input sequence. As a result, MSA can only capture small indels
and point mutations. Finding alignments that maximize homology is a computationally
difficult task; maximizing the sum of pairwise matches is NP-Hard in the number of
sequences [8–10]. Furthermore, MSA does not model evolutionary events such as inver-
sions, translocations, and the gain or loss of entire genes. In some cases, these sequences
are medically relevant and implicated in human disease [11]. Despite the inherent short-
comings, MSA is a critical first step in studying homologous relationships and is a vital
precursor to reconstructing a phylogenetic tree accurately [12]; by one estimate it is one
of the most studied scientific problems of all time [13].
Following the sequencing of the first full genome in 1995 [14], early methods attempted
to perform large-scale MSA on whole genomes. However, the initial release of the human
genome in 2000 [15] showed that the same constraints which narrow the solution space
of MSA prohibit it from capturing all of the evolutionary events that need to be tracked,
which turn out to be common [7, 16–23]. In turn, the task of aligning entire genomes and
properly accounting for and coherently reporting these events took on a separate formula-
tion known as MGA. By first finding sets of homologous regions across the input genomes
and then aligning them with MSA algorithms, MGA is able to leverage the advances in
multiple sequence alignment while still modeling genomic events that MSA does not han-
dle. Indeed, a core problem in MGA is how to correctly find these homologous regions. As
MGA consists of finding and aligning homologous regions of the input genomes suitable
for MSA, it is no surprise that MGA is inherently at least as computationally challenging
as MSA.
Two recent reviews have given a broad background on MGA and described a number
methods available for it [24, 25]. Our review will instead focus on MGA algorithms and
the implications of the growth in genome data for future development and evaluation of
MGA algorithms.

Evolutionary relationships and their impact on genome alignment

After billions of years of evolution, insertions, deletions, substitutions, and rearrange-
ments are ubiquitous to the genomes comprising the Tree of Life [26]. The accurate
identification of homologous regions is a cornerstone of comparative genomics [27] and
central to enabling the inference of their evolutionary history [28]. Homology iden-
tification refers to the identification of regions of common evolutionary history and
encapsulates the evolutionary cataloging of each nucleotide within a genome that can
span billions of nucleotides. Specifically, homologous regions can be further character-
ized as orthologs and paralogs [29, 30]. Orthologs are defined as homologous sequences
separated by a speciation event, while paralogs are those which are evolutionarily related
through a duplication event (Fig. 1). While orthologs are more likely to perform similar
functions [31], paralogs are a major force in driving evolution [32]. Inaccurate reconstruc-
tion of the underlying gene families can result in erroneous phylogenomic inferences.
Further complicating matters, genomes undergo recombination and are subject to mul-
tiple evolutionary processes that can radically alter their genomes, including inversions
Kille et al. Genome Biology (2022) 23:182 Page 3 of 22

Fig. 1 An example of evolution resulting in different classes of homology. In (a), deletion and speciation events
are depicted, resulting in both orthologs and paralogs. The resulting relationships between all pairs of X
segments as well as between all pairs of Y segments are depicted in (b). Notably, segments Yb3 and Yc2 participate
in three important homology relationship types. As the most recent common ancestor of Ya3 and Yb3 is at a
speciation event, they are orthologous to each other. Segments Yc2 and Ya1 on the other hand are paralogs, as
their homology is a result of duplication. Finally, Yb3 and Yc2 are special types of paralogs known as “false orthologs”
i.e. paralogous segments all of whose other copies in their respective genomes have since been deleted,
resulting in two segments which appear to never have been duplicated. The presence of such false orthologies
complicates the problem of core-genome alignment particularly, but also the problem of further categorizing
homologies identified through genome alignment. It is worth noting that homology relationships within a single
species’ genome do exist, but are not depicted here. For example, Ya1 , Ya2 and Ya3 are all paralogous to each other

and rearrangements. For a more in-depth discussion on the relationship between genome
position and orthology, we refer the reader to [33].
A slight variation of the problem of MGA involves aligning only the “core-genome.” The
core-genome of a set of sequences consists of orthologous segments which are shared
between all of the sequences [34, 35]. Core-genome aligners like Mauve [36] and Parsnp
Kille et al. Genome Biology (2022) 23:182 Page 4 of 22

[37] are often very scalable, as the set of relationships to consider is significantly smaller
than in MGA since they do not consider duplicates or orthologies only between a subset
of sequences. Core-genome alignment has its own unique set of challenges, though, not
least of which is that the more genomes at hand, one finds, the smaller the core-genome
gets [38].
Determining whether two homologs are orthologs or paralogs is a challenging prob-
lem which remains actively studied [39]. Additionally, the orthology relationship is
not an equivalence relationship [30], which complicates the problem of alignment.
Further complicating matters, paralogs whose duplicates have been deleted can appear as
orthologs due to the absence of duplicates.

Tracking homologous regions across assembled genomes

As MGA consists of finding and aligning homologous regions of the input genomes; a
natural first step is identifying such regions. Pairwise local sequence alignment meth-
ods have been the prevailing technique to find and track homologous segments between
genomes. First, all pairs of genomes are searched for high scoring local alignments i.e.
highly similar segments. These pairwise local alignments are then merged together to
identify regions of high similarity across subsets of the input genomes, also known as
multiple local alignments.
Pairwise local alignment tools form the foundation for the most widely used global
genome alignment methods [40, 41]. However, existing methods that identify and align
all homologous nucleotides in two or more genomes have suffered from poor scalability,
limited accuracy, or both [42]. While recent progress in the field of alignment has yielded
MSA methods that can scale up large datasets while producing accurate alignments
[43–45], these methods are “global” and therefore are less applicable when the boundaries
of the homologous regions are uncertain.
Handling local sequence similarity within global sequence alignment was previously
coined “glocal” alignment (combining global and local alignment) [46], combining local
alignments with chaining algorithms as a building block for MGA. Thus, to cope with
all of the aforementioned biological and computational hurdles, most state-of-the art
MGA methods rely on either progressive alignment [40, 41, 47] built off of “glocal” pair-
wise sequence alignments and an “aligning alignments” step [48], or divide-and-conquer
heuristics [49, 50] to scale with the rapidly increasing set of sequenced and assembled
genomes.
Over time, it has become apparent that some local alignment algorithms are bet-
ter suited for application to homology-finding in MGA than others. While using local
pairwise matches between all pairs of genomes to construct alignments that span mul-
tiple genomes is very sensitive, the number of pairwise alignments that must be run
grows quadratically with the number of genomes, so an efficient local pairwise aligner
is paramount. Alternatively, very specific local alignment methods, such as finding
sequences shared across all input genomes, are extremely efficient but only reasonable
for highly similar sequences. As we will see, the problem of MGA is expanding in all
aspects, therefore necessitating the development of new heuristics for large-scale local
alignment.
Kille et al. Genome Biology (2022) 23:182 Page 5 of 22

On the deluge and democratization of high-quality genome assemblies

Decades of striking technological development have driven huge gains in DNA sequenc-
ing throughput with parallel advances in the efficiency of algorithms to assemble the
outputs into full-length genomes. That has led to an exponential growth in public genome
databases and biologists studying all kingdoms of life are increasingly comparing ever
larger numbers of genomes, which means the problem of MGA has changed greatly in
character over that time. That being said, the development of algorithms and software for
MGA has notably not kept pace (Fig. 2).
The “thousand genome era” is here with respect to vertebrate assemblies, but with
nearly 700,000 high quality bacterial genomes available in the European Nucleotide
Archive [51] and in excess of 6 million SARS-CoV-2 genomes alone [52], it is clear that
the problem of comparing genomes comes in many shapes and sizes, each posing new
challenges for MGA (Fig. 3). In 2009, a goal was made to assemble the genomes of 10,000
different species, approximately one from each vertebrate genus [53, 54]. Encompassing
the aforementioned goal, the more recent Vertebrate Genome Project [55] seeks to obtain
a whole genome sequence of each of the over 66,000 vertebrate species. There are numer-
ous other sequencing projects, some within the Vertebrate Genomes Project such as the
Bird 10K project [56] and the sequencing of 44 ruminant genomes [57], and others for
non-vertebrate species such as the 10KP plant project [58], the Global Ant Genomics
Alliance [59], and the i5k project for arthropods [60]. The Earth BioGenome Project [61]
may even subsume them all; a moonshot started in 2018, it aims to sequence all known
eukaryotic species over 10 years. All of this is without mentioning human [62], microbial
[63–65], fungal [66], and viral [67] sequencing efforts such as the recent deluge of millions
of SARS-CoV-2 genomes [52].

Multiple genome alignment moonshot: the challenge of more genomes

Many of the challenges that hinder MGA approaches are also found in MSA and even
local alignment (Table 1) and thus, computationally feasible approaches to MGA often
leverage advances from other fields of alignment. One particular lesson from the MSA
literature is that the nature of the problem depends on several different properties of the
input data, the most obvious of which are sequence length, number of sequences, and
overall sequence similarity. For MGA, however, those three properties have particular
implications. Sequence length is an obvious difference and many MSA algorithms scale
quadratically, or approximately so, in this dimension. Since whole genomes are longer and
more structurally diverse than sequences used for MSA, virtually all MGA algorithms use

Fig. 2 Timeline of MGA tools. The original sequencing of human and mouse genomes spurred the development
of a number of multiple genome alignment tools. Following this initial spurt, the next generation of genome
aligners (starting with Enredo-Pecan and ending with Cactus) were developed, followed by a 6-year period of
silence, with Parsnp being one of the few tools released between 2012 and 2019
Kille et al. Genome Biology (2022) 23:182 Page 6 of 22

Fig. 3 a-c The number of assemblies available for different groups of eukaryotic species in NCBI. d-f The number
of eukaryotic species with available genome assemblies in NCBI. The emergence of 3rd-generation sequencing
technologies and their accompanying assembly algorithms in the early 2010s is largely responsible for the
increasing rate of novel eukaryotic genome deposits

Table 1 The many challenges of MGA and potential solutions. As MGA incorporates both local
alignment and global MSA, many challenges are shared. While some ideas from MSA have been able
to improve the capabilities of MGA, the challenges are by no means solved. Improving runtime,
performance across divergent genomes, and accuracy in the presence of repeats still remain
important challenges for MGA
Challenge Solution
Evolutionary distance between genomes increases Adding closely related species to the input dataset
alignment difficulty [68–70]. bridges the evolutionary gap between genomes in
the input and can increase alignment sizes [71].
Computational costs are currently prohibitive for Progressive methods eliminate the need for O(n2 )
many large-scale MGAs. pairwise alignments by only computing a constant
number of pairwise alignments at each of the O(n)
nodes.
Low complexity repeats often cause spurious Repeat-masking greatly simplifies the problem of
alignments and/or dramatically increase alignment. Indeed, the Cactus GitHub repository
computational cost. states that “genomes that aren’t properly masked can
still take tens of times longer to align that those that
are masked." [72]
Progressive alignments for whole genomes don’t Divide-and-conquer approaches, similar to those
account for incomplete lineage sorting or horizontal recently used for MSA, could potentially be used to
gene transfer, rendering it an incomplete model for allow sections of genomes to be treated as arising
both eukaryotes and bacteria. from different phylogenies.
Sequencing error and micro-rearrangements can Modifications to the genome graphs, similar to those
mask the existence of longer stretches of for de-Bruijn graph cleaning, can result in longer,
homologous regions. more inclusive LCBs.
The number of pairwise anchors will grow Anchoring methods that aim to identify multiple
significantly with the number of genomes being local alignments may bridge the gap between
compared and the number of anchors present in all all-pairs anchors and anchors present in all genomes.
genomes will decrease.
While MSA allows for alignment of regions within an Multiple alignment extension algorithms, such as the
LCB, there are limited methods for extending the one used in procrastAligner [73], can be employed.
borders of LCBs.
Kille et al. Genome Biology (2022) 23:182 Page 7 of 22

a two-step procedure to break the problem up: first identify highly similar regions across
two or more of the sequences known as anchors and then use these anchors to identify
larger rearrangement-free regions across input genomes known as locally colinear blocks
(LCBs).
The canonical problem statement of MSA, maximizing the number of pairwise-
matching sites, quickly becomes an intractable problem when dealing with more than a
few sequences [10] and indeed, pairwise-matching sites are often used as a metric for
MGA as well. Two immediate strategies for dealing with this are either to use a set of
pairwise alignments for which computation is tractable and resolve the inconsistencies,
or to use a heuristic to construct and optimize a local MSA directly. These two strategies
underpin the anchor recruitment approaches used in MGA algorithms, as we will see.
One heuristic method often employed is progressive alignment [74], which uses a guide
phylogeny to divide the MSA into smaller instances as well as provide an order in which
the subset MSAs are merged together. This makes the compute time scale linearly with
the number of sequences, but it also means that errors in the process will aggregate as
each sequence is added, making these methods respond poorly to more distantly related
groups of sequences without regular highly-conserved regions. Progressive alignment has
similarly been applied to MGA and as seen in Table 2, nearly half of MGA methods use

Table 2 Multiple genome alignment methods. Tools which only perform part of MGA or are only
suited for pairwise genome alignment are excluded. Progressive aligners are italicized. See [24] for a
comprehensive list of tools for all subproblems of MGA
Method Anchor type Anchor discovery Anchor chaining MSA method
method
A-Bruijn Aligner [76] Pairwise alignments BlastZ [77] Maximum Subgraph -
with Large Girth
problem [78]
Cactus [79] Pairwise alignments LastZ [80] and BAR Cactus alignment Pecan [81]
filter
M-GCAT [82] MUMs Compressed suffix Recursive match MUSCLE [44]
graph chaining
Mauve [36] MUMs GRIL [83] Greedily removes ClustalW [84]
low-scoring LCBs
MAVID [85, 86] Exon Matches BLAT [87] and Smith-Waterman [89] Needleman-Wunsch
GENSCAN [88] [90]
Mugsy [91] Pairwise alignments MUMmer [92] Min-Cut in alignment T-Coffee [93]
graph
Multi-Lagan [94] Multiple short inexact CHAOS [95] Longest increasing Sum-of-pairs/
words subsequence consensus hybrid
approach similar to
ClustalW
Multiple Genome MEMs Suffix array Maximum-weight ClustalW [84]
Aligner [96] path in an acyclic
graph [97]
Parsnp [37] MUMs Compressed suffix Weighted recursive libMUSCLE [44]
graph match chaining
Progressive Cactus Pairwise alignments LastZ [80] and BAR Cactus alignment filter Pecan [81]
[72]
progressiveMauve Extending palindromic ProcrastAligner [73] Sum-of-pairs score libMUSCLE [44]
[98] spaced seeds
SibeliaZ [99] k-mers TwoPaCo [100] Carrying paths SIMD partial order
alignment [101]
SuperMap [102] Multiple short inexact CHAOS [95] Maximum Weight Path Progressive LAGAN
words Cover [94]
TBA/MultiZ [41] Pairwise alignments BlastZ [77] - MultiZ [41]
Kille et al. Genome Biology (2022) 23:182 Page 8 of 22

some sort of progressive approach. Indeed, progressive approaches have even been used
to store MGAs [75]. To combat the issues of error propagation, progressive methods
in both MSA and MGA have adapted to share information across the subproblems and
reduce the introduction of errors [72].
For MGA, the problem of scalability in the number and diversity of genomes is exac-
erbated by the absence of the simplifying assumptions from MSA that restrict the type
of mutation events under consideration. It also makes presenting the output more chal-
lenging, as a simple row-by-row representation cannot easily display transpositions,
inversions or duplications. Methods have emerged for MSA that deal more aptly with
large numbers of more diverse sequences, for example by using a divide-and-conquer
strategy [103], but most of these have not yet been translated into analogous methods for
MGA.

Breaking down a multiple genome alignment

As noted earlier, the extreme sequence length and presence of genomic rearrangements,
gains and losses mean that nearly all MGA programs need to break the problem down
first, and can thus be separated into three parts: (1) anchoring, (2) locally colinear block
(LCB) identification, and (3) alignment, and in some cases extension, of the LCBs (Fig. 4).
In the anchor recruitment step, the goal is to find well-conserved homologous regions
between subsets of the input genomes known as anchors (Fig. 4b). These anchors serve
as building blocks to identify regions across multiple genomes which share a common
ordering of shared anchors are thus colinear and suitable for multiple sequence alignment
(Fig. 4c). In order to identify these colinear regions, the blocks are often represented in a
graph data structure which algorithms operate on to identify large locally colinear blocks.

Fig. 4 The pipeline of MGA. a An example of large-scale genomic rearrangement, insertion, and deletion
occurring across a set genomes. The lines denote bounds of homologous segments, and inversions are denoted
by crossing lines. b Anchors from the section of 3 genomes surrounded by the dotted box in a. Once again, the
lines between genomes represent homology. The labeled blocks on each genome correspond to anchors, where
two blocks with the same label are inferred to be potentially homologous sites. c The alignment graph obtained
by merging the 3 linear genome graphs in b. At this step, the aim of MGA is to find colinear paths in the graph i.e.
sequences of anchors which are traversed by a group of genomes in the same order. d Often times, the initial set
of anchors will be too noisy, containing spurious alignments which prevent the formation of longer, more reliable
colinear paths. By removing anchor F, the alignment graph becomes much simpler and yields longer colinear
paths, where each set of colinear paths is denoted by the color. e For each colinear path, MGA tools perform an
MSA, yielding a set of sequence alignments which together make up the genome alignment
Kille et al. Genome Biology (2022) 23:182 Page 9 of 22

These blocks are then passed to the final step of MSA and the resulting set of MSAs
constitutes the MGA (Fig. 4d). In order to imagine how MGA might begin to cope with
more genomes it is helpful to consider the effects of scaling on each of the first two steps
separately and in detail.
In the following sections, we will review different approaches to (1) and (2). In many
cases, these steps are modular. For example, a recent study took the popular MGA
program Mugsy [91] and replaced the MUMmer [92] anchoring step with filtered space
word matches [104], yielding a new alignment pipeline which had significantly improved
results [105].

Anchor recruitment
Most anchoring recruitment algorithms can be classified in two distinct ways: pairwise
versus multiple anchors, and exact versus approximate. The first classification is self-
explanatory, whether the recruitment identifies anchors that link regions in two genomes
at a time or anchors that jointly link at least two of the input genomes directly. The trade-
off is that while pairwise anchoring is more sensitive, computing all pairwise alignments
scales quadratically with the number of genomes. The second classification is whether
exact matching or something more approximate like gapped alignment is used for the
anchoring. Again, the trade-off between the two approaches is speed versus sensitivity.
Importantly though, sensitivity is a double-edged sword in MGA. Any anchoring algo-
rithm that emphasizes multiple-genome anchors that span large subsets of the input set
of genomes is going to lose sensitivity as the number of genomes and their diversity
increases. On the other hand, the increased sensitivity of pairwise alignment is often
accompanied by more spurious alignments.

Pairwise-Exact: MUMmer
The MUMmer suite is a set of tools for pairwise genome alignment [92, 106–108]. The
mummer tool from the suite works by first finding all maximal exact matches (MEMs)
between a reference genome and a query genome longer than some minimum length l by
using a suffix array [109]. MEMs which are not unique in either genome can be imprecise
for determining homology and also can lead to a quadratic explosion of alignments for
high copy number repeats. To accommodate this, MUMmer discards all matches which
are not unique in the reference by default.
The MUMmer suite also contains a pairwise aligner, nucmer, which uses the intuition
that matches which are close together and appear in the same order in both genomes
often make up a rearrangement-free homology. First, all matches within distance g of
each other are clustered together. From each cluster, the maximum length colinear block
of matches is extracted. If the total number of nucleotides from all matches within an
extracted colinear block does not add up to some parameter c, the block is discarded.
For each remaining colinear block, the regions between matches as well as the flanking
regions of the block are aligned using the Smith-Waterman alignment algorithm [89].

Pairwise-Approximate: LastZ
Another widely accepted methodology for computing pairwise genome alignments is the
BlastZ method, introduced by PipMaker [110] in 2002 and later improved upon in 2003
[77] and again with its successor LastZ [80]. In order to obtain the anchors, LastZ follows
a similar methodology to that laid out by gapped Blast [111], namely by using the X-drop
Kille et al. Genome Biology (2022) 23:182 Page 10 of 22

method [112] to compute efficient gapped and gapless extensions from seeds. Given a
type of seed, of which LastZ natively supports a few, LastZ finds all pairs of matching seeds
across the two genomes. For each pair, a gapped extension is computed, and all extensions
scoring below some threshold are discarded, leaving only the high scoring pairs (HSPs).
An optional additional step computes a more sensitive seed-and-extend step between
colinear anchors.

Multiple-Exact: Parsnp
Parsnp discovers anchors for MGA by using a compressed suffix graph (CSG) to obtain
maximal unique matches (MUMs) that are present in all input genomes [37]. Parsnp is
thus particularly suited for closely related genomes, as the number of MUMs drops off as
the sequences diverge. Even one highly divergent sequence is enough to critically reduce
the number of MUMs across the entire input and as a result Parsnp provides a first pass
filtering step in which any significantly divergent genomes from a user defined reference
are discarded.
Following the filtering step, Parsnp constructs a CSG of the reference, a data struc-
ture which is able to perform the same MUM finding capabilities of a suffix tree but in a
significantly more compressed representation. It should be noted that excluding the fil-
tering step, the selection of the reference will have no effect on the output produced. Each
input genome is then streamed through the CSG, resulting in a candidate set of pairwise
MUMs. The final set of MUMs used as anchors is the intersection of MUMs obtained
from each input genome.

Multiple-Approximate: ProcrastAligner
Using the seed-and-extend strategy, ProcrastAligner produces local multiple alignments
in a manner which aims to find anchors which span as many of the input genomes
as possible. First, ProcrastAligner indexes the location of a set of palindromic spaced
seed [113] patterns in order to match both strands simultaneously. ProcrastAligner ini-
tiates a priority queue ordered on the multiplicity of each seed. The extension step
takes seeds from the queue and computes a gapped extension from each side until a
gap of length w is incurred. Upon termination of an extension, it is linked to nearby
matches. This way, when a less frequent match is popped from the queue, it can
avoid computing extensions of linked neighbor regions, as they have already been
aligned.

Graph data structures for alignment

Many alignment algorithms represent the anchors and the sequences between them in
graph data structures. Common alignment errors such as short spurious alignments
are often signaled by motifs in the graph such as short cycles. By constructing the
graph data structure for an alignment, algorithms can identify these motifs and attempt
to correct them. Additionally, graph representations provide a straightforward way to
both visualize alignments as well as compute them in parallel. In order to describe
the locally colinear block construction methods, it is necessary to briefly describe
the different graph data-structures that these algorithms operate on. We refer the
reader to [114] for a more in-depth discussion of graph data structures for genome
alignment.
Kille et al. Genome Biology (2022) 23:182 Page 11 of 22

Basic alignment graphs

In the basic alignment graph [10], each node is a genomic segment. For each pair of
aligned segments there is an undirected block-edge and for each pair of adjacent segments
there is a directed adjacency-edge. A block is a block-edge connected component in the
alignment graph. These graphs often serve as the basis for construction of other types of
genomic graph data structures.

A-Bruijn graphs
In 2004, Pevzner et al. presented the concept of an A-Bruijn graph [78], which merges all
nodes in a block-edge connected component in the alignment graph into one single node.
While A-Bruijn graphs require additional labeling to recover the original genomes, they
have the advantage of representing local multiple alignments with a single node.

Enredo graphs
Enredo graphs were introduced as part of the Enredo-Pecan aligner [115] and are similar
to A-Bruijn graphs, with the addition that they are able to represent relative orientation
of blocks. Let G = (V , E) be an A-Bruijn graph and G = (V  , E ) be the corresponding
Enredo graph. The definition of V  is straightforward, we split every vertex in the A-
Bruijn graph into a head and tail vertex: V  = {vh , vt |v ∈ V }. For each pair of head and tail
vertices for a vertex from the A-Bruijn graph, we create a directed block-edge (vt , vh ), and
the set of adjacency-edges for the Enredo graph is Ea = {(ut , vh )|(u, v) ∈ E}. By adding
these head and tail vertices, Enredo graphs are able to provide a sense of orientation to
the local alignments from the A-Bruijn graph.

Cactus graphs
The Cactus aligner [79] uses a more sophisticated class of graphs, cactus graphs [116],
which have the property that every edge is part of at most one simple cycle. We refer the
reader to [79, 117] for a more detailed discussion of cactus graphs, but will describe an
outline here to provide context for the Cactus LCB construction step. In a cactus graph,
the edges are block edges and the nodes in the cactus graph are nets, which contain the
adjacencies between the connected edges. A fundamental cycle in the cactus graph is
therefore a series of consecutive blocks, or local alignments. Additionally, by recursively
constructing cactus graphs from the adjacencies within nodes, cactus graphs can easily
represent different resolutions of genome alignment.

de-Bruijn graphs
For a genome g and some integer k, we denote de-Bruijn graph [118] as G(g, k). The nodes
of G(g, k) are all k-mers in g, and for every pair of vertices u and v, there is a directed edge
u → v if the k − 1 length suffix of u is the same as the k − 1 length prefix of v. Therefore,
edges in the graph represent (k + 1)-mers. A de-Bruijn graph of multiple sequences is the
union of the de-Bruijn graphs for each individual sequence.

Locally colinear block construction

Given a set of anchors represented as a graph, the next step is to identify locally colinear
blocks (LCBs), i.e. regions which share a common ordering of anchors. While the ini-
tial set of anchors are sufficient to construct LCBs, they are often noisy and may contain
Kille et al. Genome Biology (2022) 23:182 Page 12 of 22

artifacts of micro-rearrangements and assembly error. Consequently, the focus of this

step is removing anchors in order to obtain longer, more reliable LCBs. Often times,
once the LCBs are obtained methods will repeat the anchoring and LCB construction
steps on the regions in-between colinear anchors within an LCB, as these regions are
likely to be homologous as well given the high confidence of their flanking regions. In the
this section, we provide examples of LCB construction algorithms for different anchor
representations.

Graph-free LCB construction: ProgressiveMauve

ProgressiveMauve [98] uses MUMs for anchoring, similar to Mauve [36] and Parsnp [37].
However, in ProgressiveMauve the MUMs only need to be present in at least two genomes
as opposed to every genome. In order to compute LCBs from this anchoring, Progres-
siveMauve works in a pairwise progressive fashion, iteratively working up a guide tree of
all genomes. This way, a pairwise alignment only needs to consider MUMs between both
genomes. For an iteration of the progressive alignment, ProgressiveMauve processes a
node whose two children in the guide tree have been processed. The aim of each iteration
is to remove some subset of anchors to maximize a sum-of-pairs score, where the set of
pairs is the cartesian product of the descendants from each of the children of the current
node. The score for each pair takes into account the number of LCBs shared between the
pair as well as the score of the local alignments that make up the LCB. After identifica-
tion of the LCBs, ProgressiveMauve uses the gapped multiple alignment extension from
procrastAligner [73] in order to extend the boundaries of identified LCBs.

A-Bruijn graphs: Mugsy

Mugsy [91] represents the initial set of anchors using a basic alignment graph, with the
additional feature that it represents alignment orientation in order to capture inversions.
Since repeats and duplications can result in multiple segments from anywhere in the same
genome being part of the same connected component in an alignment graph, Mugsy uses
a greedy depth-first search on each component in order to compute how anchors will
be recruited in order to construct the A-Bruijn graph. When the search comes across
a duplication or repeat that is not within some predefined distance of its homolog, the
copy becomes a new anchor. The resulting set of connected components in the alignment
graph is used to construct an “anchor graph,” which shares many of the same features
of the A-Bruijn graph. The initial set of LCBs is then calculated by finding simple paths
which do not pass through vertices with degree larger than two.
In order to extend the LCBs, Mugsy attempts to remove breakpoints that are artifacts
of micro-rearrangements or the initial greedy selection of anchors. To address the former
issue, LCBs shorter than some threshold L are removed from the graph and any neces-
sary adjacency edges are introduced in their place. Any LCB in the graph containing two
anchors a and b separated by more than G from the same genome are disconnected. Such
a disconnection is made by using a and b as source and sink nodes in a min-cut instance
[119] on the graph where each edge is weighted by the number of genomes that share that
adjacency edge.

Enredo graphs: Enredo-Pecan aligner

The Enredo algorithm for creating LCBs in the Enredo graph contains three major steps
[115]. In the first, paths in the graph which pass through similar anchor points are merged
Kille et al. Genome Biology (2022) 23:182 Page 13 of 22

into single paths which represent the union of the represented alignments. This way, iso-
lated cases of missing or inserted anchors won’t disrupt the ability to construct longer
LCBs.
In the second step, Enredo attempts to remove small spurious matches which may result
in a scenarios where two potential disjoint LCBs intersect at a spurious anchor. By looking
for local multiple alignments whose partitioning into two disjoint alignments results in
longer LCBs, the occurrence of such false homologies can be reduced. As a final step,
small cycles in the graph, often a result a result of assembly error and duplication, are
identified and short anchors from the cycle whose removal results in a larger chain are
deleted.

Cactus graphs: Cactus aligner

Given a cactus graph, the goal is once again to remove a subset of local alignments in
order to improve the obtained LCBs. Cactus provides a mathematical formulation of
this problem in terms of the cactus graph. The Maximum weight Cactus subgraph with
large chains (MSLC) problem is an optimization problem which seeks to remove as few
local alignments as possible while ensuring that all chains in the cactus graph contain
at least α alignment columns in total [79]. The cactus aligner uses a greedy approach,
named the Cactus Alignment Filter, to solve this problem by iteratively removing local
alignments from the graph to increase the size of small chains and then adding back in
alignments between colinear blocks in a chain. By increasing the cutoff of the minimum
chain size at each iteration, the algorithm works up to a graph in which all chains are
longer than α.
Within a net of the cactus graph, it is likely that there are still rearrangements, albeit
smaller, among the adjacencies. In order to resolve these rearrangements, the Cactus
aligner seeks to construct another cactus graph from the set of adjacencies within a net.
To do this, Cactus uses the Base-level Alignment Refinement algorithm (BAR). BAR uses
a modification of the Pecan aligner [115] to align adjacencies within a net that share an
endpoint. This set of alignments is likely to contain spurious matches, and therefore the
BAR algorithm refines these alignments in order to increase a score determined by a pair-
wise HMM [120] as well as create consistent alignments. The resulting alignments from
the BAR algorithm are then used to construct a cactus graph on the net. This recursion
continues and results in a multi-level cactus graph.

De-Bruijn graphs: SibeliaZ

SibeliaZ [99] takes a similar approach to its predecessor, Sibelia [121], by first construct-
ing a de-Bruijn graph of the concatenated genomes using TwoPaCo [100], a tool designed
specifically for constructing compacted de-Bruijn graphs from many large genomes.
With the de-Bruijn graph in hand, SibeliaZ constructs LCBs by iteratively extracting
“carrier paths” in the graph. These carrier paths are constructed by starting from a ran-
dom edge in the graph and iteratively following the heaviest unvisited edge, where the
weight of an edge is the number of genomes that it represents. The intuition behind
this is that LCBs should share a significant number of k-mers, and therefore carrying
paths should be able to represent LCBs in the de-Bruijn graph. These walks start at
random edges and are terminated when either the score of the path falls below some
threshold or there are no valid steps left. The score of a carrying path in SibeliaZ
Kille et al. Genome Biology (2022) 23:182 Page 14 of 22

reflects how well the carrying path aligns with the segments it represents, and a car-
rying path represents a segments if it overlaps with at least m nucleotides from that
segment.

Future perspectives
The significant increase in quality and quantity of assemblies has direct consequences for
the characteristics of MGA. Long-read sequencing has enabled the closing of countless
gaps as well as the correct assembly of repeated regions, which logically leads to more
accurate genome alignments. Where short read assembly had over-collapsed and under-
collapsed regions, MGA methods would subsequently make erroneous calls of deletions
and duplications, respectively. Historically, many methods addressed such errors by iden-
tifying and removing small local alignments that correspond to micro-rearrangements. In
addition to resolving repeats, complete genomes will also provide adjacency edges along
with their sequence labels to alignment graphs which were previously absent or unlabeled.
The era of complete genomes also unleashes the possibility of correctly aligning sequences
which often elude current methods of analysis. These alignments, both between humans
as well as between humans and model-species, can potentially yield new insights into
structural variation and human disease [122].
With genomes now being complete and repeat regions resolved correctly, MGA algo-
rithms can focus more on finding accurate local alignments and untangling the resulting
net of anchors and less on adjusting for sequencing error. As MGA is a broad field
encompassing repeat-masking, local alignment, graph theory, LCB construction, and
MSA, it is no surprise that there are a number of avenues for future improvements. In
this section, we focus on five main areas that we believe will be key to unlocking future
advances in MGA.

Area 1: Validation and benchmarks

In order to improve the performance of MGA methods, it is critical to develop stan-
dardized validation methods which accurately model the problem at hand. The lack of
a comprehensive benchmark for MGA methods spurred the Alignathon [123] in 2014.
In an effort to evaluate existing methods and provide a usable benchmark, the authors
used EVOLVER [124] to simulate two datasets, one representing a closely related set
of 4 primates and the other representing a more divergent set of 5 mammals. Methods
were evaluated by considering the site level, pairwise precision, and recall of the esti-
mated alignments, but only on a sample of sub-regions of the whole. The Alignathon was
a high-quality study and the first of its kind, but the small number of genomes involved
has meant diminishing applicability as the availability of data grows. Moreover, an MGA
algorithm not only has to be accurate within LCBs at the site level, it must also consider
challenges like the extent of the alignment identified and whether it captures the more
exotic mutations like translocations and duplications.
Further development of a broader benchmark and a more comprehensive set of eval-
uation metrics would be useful as new tools are developed for the volume of data now
common in MGA. With the new era of T2T genomes comes much higher quality assem-
blies. Indeed, it may now be worth regenerating the alignathon simulation datasets from
the now completed T2T human genome [2, 125], although the most recent whole-
genome simulation method, ALF [126], is nearly a decade old. In order to provide a better
Kille et al. Genome Biology (2022) 23:182 Page 15 of 22

benchmark for MGA, new genome-wide evolution simulation approaches may also be
necessary. Parsnp [37] and SibeliaZ [99] both simulated a bacterial dataset for bench-
marking, but there is still no accepted benchmark for aligning thousands of bacterial
genomes.
Finally, a direction for validation and benchmarking that may be appropriate is to
depart from the paradigm of evaluating by measuring quantitative accuracy with respect
to a ground truth. An analogy is the N50 metric in genome assembly, which is informa-
tive about a method’s output but is not properly a higher-is-better measure of accuracy
(though it is sometimes liberally applied as such). For MGA, given a biological bench-
mark of appropriate size such as the over 130 high-quality assemblies already available
in the Vertebrate Genomes Project [55], some measures of interest would be running
time and fraction of overall genome content aligned in LCBs with at least 90% aver-
age nucleotide identity (or a different cutoff if appropriate). These alone would be useful
points of comparison for a user approaching an application, and they are likely not the
only ones.

Area 2: Reducing computational requirements

Due to the quadratic complexity of all-pairs anchoring methods, a priority aim of
many MGA tools is to reduce the computational cost of anchoring while still main-
taining a high level of sensitivity. The utilization of a progressive alignment in Cactus
has proved to do just that, enabling an alignment of over 600 amniotes consisting
of over 1 trillion base pairs. While the progressive approach of Progressive Cac-
tus lends itself to a linear time complexity with respect to the number of input
genomes, it is unclear how much computational resources this alignment required.
In fact, the authors of SibeliaZ showed that Progressive Cactus was unable to com-
plete an alignment on 8 mouse genomes from a recent dataset [127] in 1 week on
32 threads [99].
With the advent of extremely powerful GPUs in recent decades has also come GPU
adaptations to many alignment algorithms [128–130]. Notably, both MUMmer and LastZ
have been fitted for GPU use [131, 132], with the latter improvement yielding a consistent
13x speedup on a vertebrate dataset. While GPUs and large compute clusters, whose use
has been made easier by workflow engines such as Toil [133] and Nextflow [134], may
provide significant wall-clock time speedups in proportion to the resources available, they
are only beneficial to those with the necessary resources. In addition, the speedups may
be drowned out by the thousands of genomes to come. It follows that hardware-agnostic
algorithmic improvements will be necessary in order to enable accurate alignments of
thousands of large genomes.

Area 3: Improved anchoring

Worthy of its own review, the seed-and-extend approach to local alignment has been
shown to be heavily influenced by the type of seed used. LAST [135], a pairwise sequence
aligner, showed that seeds which are chosen based on rareness as opposed to length
leads to the number of matches increasing linearly with sequence length as opposed to
quadratically.
Both MashMap [136, 137] and MiniMap [138, 139] use minimizers [140] in order to
select seeds for pairwise genome alignment, but both algorithms are tailored towards
Kille et al. Genome Biology (2022) 23:182 Page 16 of 22

mapping long reads to references, as opposed to finding small alignments across diver-
gent genomes which are suitable for anchors. It remains to be seen whether min-
imizers will break into the space of general MGA. Strobemers [141] and syncmers
[142], two recent methods for seeding which aim to be more sensitive than mini-
mizers while still being more sparse than static seeding approaches, are also promis-
ing avenues for improving genome anchoring. An additional area of improvement
is based on the idea of designing seeds beforehand or allowing seed characteris-
tics to be dynamic, as opposed to selecting an arbitrary set based off a seed rule
[135, 143, 144].
As seeding is an approach that is used as a tool for more specific applications, it is
necessary to benchmark the seed types for different contexts. For example, Edgar [142]
substituted syncmers into both MiniMap2 [139] and Kraken [145] and compared the
results. It would be interesting to see a similar but more comprehensive benchmark
performed for seeding methods in MGA.

Area 4: Optimizing LCB construction

It could be argued that the method of representing the graph genome, modifying it, and
identifying LCBs in the graph is the core novelty of most modern MGA approaches. Given
the prominence of genomic rearrangements and repeats in most genomes, the problem of
finding homologous regions boils down to untangling a set of more clear and conserved
anchoring regions. There are two likely ways forward in order to increase MGA align-
ment quality given a set of anchors. The first is to come up with new representations of
sequences and their anchors. This has been an approach which has yielded significant
advances in MGA, with each of the graph types discussed in this review contributing to
major milestones of MGA.
Additionally, LCB construction can be improved by improving algorithms for existing
representations of genomes and their anchors. Often, methods for removing anchors to
construct better LCBs are posed as mathematical optimization problems. In some cases,
such as Mugsy [91], these problems are known to be solvable in polynomial time. For
other algorithms, such as Cactus’ MSLC problem [79], the A-Bruijn aligner’s MSLG prob-
lem [78], and ProgressiveMauve’s sum-of-pairs optimization [98], there are no known
polynomial time optimal solutions and therefore heuristic algorithms are employed. The-
oretical guarantees, or at the very least heuristic improvements, are likely to advance the
field as well.

Area 5: Reticulation-aware MGA

Progressive approaches to MSA and MGA have been largely successful in enabling the
alignment of larger sets of sequences. That being said, the progressive method it is not
without faults of its own. Sophisticated progressive methods which account for global
data are still susceptible to propagating early errors up the tree [146]. Additionally,
progressive methods require a guide tree which is often calculated with pairwise dis-
tances. It has been shown that reordering sequences with very similar distances can
result in MSA instability [147]. This instability can be remedied at the cost of runtime
by allowing multiple alignments at internal nodes as opposed to strictly pairwise (as
in Progressive Cactus). It is also worth noting that most progressive alignment meth-
ods assume that all areas of the genome share the same phylogeny and are free of gene
Kille et al. Genome Biology (2022) 23:182 Page 17 of 22

flow, both of which is known to be a significant factor in evolution [148–150]. Unfor-

tunately, most studies of the effects of progressive alignment are restricted to MSA. It
remains to be studied how these factors impact the performance of progressive methods
on MGA.
One intersection between the problem of MGA and MSA is the representation of inter-
nal nodes. While using ancestral reconstruction algorithms to replace internal nodes by
single sequences is simpler and often more efficient, it has been shown that different
methods of MSA can lead to biases in ancestral reconstruction [151]. Another method
to solve the subproblems of progressive alignment is by representing internal nodes as
alignment profiles. Profile alignment in MSA is an old and well studied problem [152]
but is significantly more complex in MGA as the profiles should ideally be able to cap-
ture genomic rearrangements and large indels as well. Recent genome alignment methods
have shown the promise of representing and aligning genome profiles, and more advances
are likely to follow [153, 154].
Future methods for MGA will likely follow the path laid out for them by years of
work on progressive and divide-and-conquer approaches to MSA. By horizontally split-
ting the input genomes up, different regions may be treated with different phylogenies.
More sophisticated representations which allow for gene flow may also contribute to cor-
rectly identifying and aligning shared regions of distal genomes. Furthermore, a divide
and conquer approach similar to SATe [103] may aim to split up the input sequences
and iteratively co-estimate and refine the alignments and rearrangements along with
the phylogeny, yielding more accurate genome alignments as well as more insightful
phylogenies.

Conclusion
There is a stark contrast between the number of MGA approaches developed in the first
decade of the 2000s as opposed to the last 10 years. Indeed, there have only been a
few methods published since the Alignathon that comprehensively tackle MGA, notably
SibeliaZ and Cactus. It is likely that the increasing arrival rate of high quality assemblies
will rejuvenate the effort towards advances in MGA, whether it be improvements to exist-
ing methods, new representations of anchors, or novel approaches to the problem as a
whole. It is entirely possible the computational methods that are in large part responsible
for the deluge of T2T assemblies will help to inspire a new generation of MGA approaches
to decipher their evolutionary histories. Regardless, future improvements will be required
to help advance MGA and allow it to scale up with the vast number of genomes being
ushered in by the T2T era.

Supplementary Information
The online version contains supplementary material available at https://doi.org/10.1186/s13059-022-02735-6.

Additional file 1. Review history.

Acknowledgements
We would like to thank Adam Phillippy and Aaron Darling for numerous insightful discussions on multiple genome
alignment over the past several years. We would also like to thank the reviewers for their constructive and thorough
feedback.

Peer review information

Andrew Cosgrove was the primary editor of this article and managed its editorial process and peer review in
collaboration with the rest of the editorial team.
Kille et al. Genome Biology (2022) 23:182 Page 18 of 22

Review history
The review history is available as Additional file 1.

Authors’ contributions
B.K. and T.J. led the project. B.K., A.B., F.S, M.N., and T.J. wrote, reviewed, and edited the manuscript. All authors approved
the final manuscript.

Authors’ Twitter handles

@traingene (Todd Treangen), @BKille (Bryce Kille), @AdvaitBalaji (Advait Balaji), @sedlazeck (Fritz Sedlazeck),
@michaelnute (Michael Nute)

Funding
B.K. was funded by a fellowship from the National Library of Medicine Training Program in Biomedical Informatics and
Data Science (5T15LM007093-30, PI: Kavraki). A.B., B.K., and T.T. were supported in part by NIH grant P01-AI15299901
supported by National Institute of Allergy and Infectious Diseases (NIAID). T.T. was supported in part by Centers for
Disease Control (CDC) contract 75D30121C11180. M.N. was funded by a fellowship from the National Library of Medicine
Training Program in Biomedical Informatics and Data Science (T15LM007093, PI: Kavraki). F.S. was supported in part by
ASAP-000430. T.T. was also supported by National Science Foundation (NSF) grant EF-2126387.

Availability of data and materials

Data for Fig. 3 was obtain from the taxa-specific assembly_summary.txt files located at https://ftp.ncbi.nlm.nih.
gov/genomes/genbank.

Declarations
Ethics approval and consent to participate
Not applicable.

Consent for publication

Not applicable.

Competing interests
The authors declare that they have no competing interests.

Received: 20 October 2021 Accepted: 21 July 2022

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