molecular phylogenetics Molecular phylogenetics () is the branch of phylogeny that analyzes genetic, hereditary molecular differences, predominantly in DNA sequences, to gain information on an organism's evolutionary relationships. From these analyses, it is possible to ...

, relationships among individuals are determined using character traits, such as DNA,

RNA Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...

protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...

, which may be obtained using a variety of

sequencing In genetics and biochemistry, sequencing means to determine the primary structure (sometimes incorrectly called the primary sequence) of an unbranched biopolymer. Sequencing results in a symbolic linear depiction known as a sequence which succ ...

technologies. High-throughput

next-generation sequencing Massive parallel sequencing or massively parallel sequencing is any of several high-throughput approaches to DNA sequencing using the concept of massively parallel processing; it is also called next-generation sequencing (NGS) or second-generation s ...

has become a popular technique in transcriptomics, which represent a snapshot of gene expression. In

eukaryote Eukaryotes () are organisms whose cells have a nucleus. All animals, plants, fungi, and many unicellular organisms, are Eukaryotes. They belong to the group of organisms Eukaryota or Eukarya, which is one of the three domains of life. Bacte ...

s, making phylogenetic inferences using

is complicated by

alternative splicing Alternative splicing, or alternative RNA splicing, or differential splicing, is an alternative splicing process during gene expression that allows a single gene to code for multiple proteins. In this process, particular exons of a gene may be ...

, which produces multiple transcripts from a single

gene In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a ba ...

. As such, a variety of approaches may be used to improve phylogenetic inference using transcriptomic data obtained from

RNA-Seq RNA-Seq (named as an abbreviation of RNA sequencing) is a sequencing technique which uses next-generation sequencing (NGS) to reveal the presence and quantity of RNA in a biological sample at a given moment, analyzing the continuously changing c ...

and processed using computational phylogenetics.

Sequence acquisition

There have been several transcriptomics technologies used to gather sequence information on

transcriptome The transcriptome is the set of all RNA transcripts, including coding and non-coding, in an individual or a population of cells. The term can also sometimes be used to refer to all RNAs, or just mRNA, depending on the particular experiment. The t ...

s. However the most widely used is

RNA-Seq

RNA reads may be obtained using a variety of RNA-seq methods.

Public databases

There are a number of public databases that contain freely available RNA-Seq data.

Assembly

Sequence assembly

RNA-Seq data may be directly assembled into transcripts using sequence assembly. Two main categories of sequence assembly are often distinguished: # ''de novo'' transcriptome assembly - especially important when a

reference genome A reference genome (also known as a reference assembly) is a digital nucleic acid sequence database, assembled by scientists as a representative example of the set of genes in one idealized individual organism of a species. As they are assemble ...

is not available for a given

species In biology, a species is the basic unit of classification and a taxonomic rank of an organism, as well as a unit of biodiversity. A species is often defined as the largest group of organisms in which any two individuals of the appropriate s ...

. # Genome-guided assembly (sometimes mapping or reference-guided assembly) - is capable of using a pre-existing reference to guide the assembly of transcripts Both methods attempt to generate biologically representative isoform-level constructs from RNA-seq data and generally attempt to associate isoforms with a gene-level construct. However, proper identification of gene-level constructs may be complicated by recent duplications, paralogs,

or gene fusions. These complications may also cause downstream issues during ortholog inference. When selecting or generating sequence data, it is also vital to consider the tissue type, developmental stage and environmental conditions of the organisms. Since the

represents a snapshot of

gene expression Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, protein or non-coding RNA, and ultimately affect a phenotype, as the final effect. The ...

, minor changes to these conditions may significantly affect which transcripts are expressed. This may detrimentally affect downstream ortholog detection.

Public databases

RNA may also be acquired from public databases, such as GenBank, RefSeq
1000 Plants (1KP)
an
1KITE
Public databases potentially offer curated sequences which can improve inference quality and avoid the computational overhead associated with sequence assembly.

Inferring gene pair orthology/paralogy

Approaches

Orthology or

paralogy Sequence homology is the biological homology between DNA, RNA, or protein sequences, defined in terms of shared ancestry in the evolutionary history of life. Two segments of DNA can have shared ancestry because of three phenomena: either a spec ...

inference requires an assessment of

sequence homology Sequence homology is the biological homology between DNA, RNA, or protein sequences, defined in terms of shared ancestry in the evolutionary history of life. Two segments of DNA can have shared ancestry because of three phenomena: either a spe ...

, usually via

sequence alignment In bioinformatics, a sequence alignment is a way of arranging the sequences of DNA, RNA, or protein to identify regions of similarity that may be a consequence of functional, structural, or evolutionary relationships between the sequences. Alig ...

. Phylogenetic analyses and

are often considered jointly, as phylogenetic analyses using DNA or

require sequence alignment and alignments themselves often represent some hypothesis of

homology Homology may refer to: Sciences Biology *Homology (biology), any characteristic of biological organisms that is derived from a common ancestor * Sequence homology, biological homology between DNA, RNA, or protein sequences *Homologous chrom ...

. As proper ortholog identification is pivotal to phylogenetic analyses, there are a variety of methods available to infer orthologs and paralogs. These methods are generally distinguished as either graph-based algorithms or tree-based algorithms. Some examples of graph-based methods include InParanoid, MultiParanoid, OrthoMCL, HomoloGene and OMA. Tree-based algorithms include programs such as OrthologID or RIO. A variety of BLAST methods are often used to detect orthologs between

as a part of graph-based algorithms, such as MegaBLAST, BLASTALL, or other forms of all-versus-all BLAST and may be

nucleotide Nucleotides are organic molecules consisting of a nucleoside and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecules wi ...

- or

-based alignments. RevTrans will even use protein data to inform DNA alignments, which can be beneficial for resolving more distant phylogenetic relationships. These approaches often assume that best-reciprocal-hits passing some threshold metric(s), such as identity, E-value, or percent alignment, represent orthologs and may be confounded by incomplete lineage sorting.

Databases and tools

It is important to note that orthology relationships in public databases typically represent gene-level orthology and do not provide information concerning conserved alternative splice variants. Databases that contain and/or detect orthologous relationships include:
DIOPT

Ensembl Compara

GreenPhylDB

HaMStR

HomoloGene

InParanoid

MultiParanoid

OMA
*

OrthoDB OrthoDB presents a catalog of orthologous protein-coding genes across vertebrates, arthropods, fungi, plants, and bacteria. Orthology refers to the last common ancestor of the species under consideration, and thus OrthoDB explicitly delineates or ...

OrthoFinder OrthoFinder is a Command-line_interface, command-line software tool for Bioinformatics#Comparative_genomics, comparative genomics. OrthoFinder determines the correspondence between genes in different organisms (also known as orthology analysis ...

OrthologID

OrthoMCL

OrtholugeDB

PhylomeDB

TreeFam

eggNOG

metaPhOrs

Multiple sequence alignment

As eukaryotic transcription is a complex process by which multiple transcripts may be generated from a single

through

with variable expression, the utilization of RNA is more complicated than DNA. However,

transcriptomes The transcriptome is the set of all RNA transcripts, including coding and non-coding, in an individual or a population of cells. The term can also sometimes be used to refer to all RNAs, or just mRNA, depending on the particular experiment. The t ...

are cheaper to sequence than complete genomes and may be obtained without the use of a pre-existing

. It is not uncommon to

translate Translation is the communication of the meaning of a source-language text by means of an equivalent target-language text. The English language draws a terminological distinction (which does not exist in every language) between ''transl ...

RNA sequence into protein sequence when using transcriptomic data, especially when analyzing highly diverged taxa. This is an intuitive step as many (but not all) transcripts are expected to code for protein isoforms. Potential benefits include the reduction of mutational biases and a reduced number of characters, which may speed analyses. However, this reduction in characters may also result in the loss of potentially informative characters. There are a number of tools available for multiple sequence alignment. All of which possess their own strengths and weaknesses and may be specialized for distinct sequence types (DNA, RNA or protein). As such, a splice-aware aligner may be ideal for aligning RNA sequences, whereas an aligner that considers protein structure or residue substitution rates may be preferable for translated RNA sequence data.

Opportunities and limitations

Using RNA for phylogenetic analysis comes with its own unique set of strengths and weaknesses.

Advantages

* large set of characters * cost-effective * not dependent upon a

Disadvantages

* expenses of extensive taxon sampling * difficulty in identification of full-length, single-copy transcripts and orthologs * potential misassembly of transcripts (especially when duplicates are present) * missing data as a product of the transcriptome representing a snapshot of expression or incomplete lineage sorting

Sequence acquisition

RNA-Seq

Public databases

Assembly

Sequence assembly

Public databases

Inferring gene pair orthology/paralogy

Approaches

Databases and tools

Multiple sequence alignment

Opportunities and limitations

Advantages

Disadvantages

See also

References

External links