Codon usage patterns in Nematoda: analysis based on over 25 million codons in thirty-two species

gb-2006-7-8-r75 GBJ Research Codon usage patterns in Nematoda: analysis based on over 25 million codons in thirty-two species Mitreva Makedonka mmitreva@watson.wustl.edu Wendl C Michael mwendl@watson.wustl.edu Martin John jmartin@watson.wustl.edu Wylie Todd twylie@watson.wustl.edu Yin Yong yyin@watson.wustl.edu Larson Allan larsontl@biology.wustl.edu Parkinson John jparkin@sickkids.ca Waterston H Robert waterston@gs.washington.edu McCarter P James jmccarte@watson.wustl.edu

Genome Sequencing Center, Washington University School of Medicine, St Louis, Missouri 63108, USA

Department of Biology, Washington University, St. Louis, Missouri 63130, USA

Hospital for Sick Children, Toronto, and Departments of Biochemistry/Medical Genetics and Microbiology, University of Toronto, M5G 1X8, Canada

Department of Genome Sciences, University of Washington, Seattle, Washington 98195, USA

Divergence Inc., St Louis, Missouri 63141, USA

Genome Biology 1465-6906 2006 7 8 R75 http://genomebiology.com/2006/7/8/R75 16907990 10.1186/gb-2006-7-8-r75 20 4 2006 30 6 2006 14 8 2006 14 08 2006 2006 Mitreva et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Codon usage in worms

A codon usage table for 32 nematode species is presented and suggests that total genomic GC content drives codon usage.

Abstract

Background

Codon usage has direct utility in molecular characterization of species and is also a marker for molecular evolution. To understand codon usage within the diverse phylum Nematoda, we analyzed a total of 265,494 expressed sequence tags (ESTs) from 30 nematode species. The full genomes of Caenorhabditis elegans and C. briggsae were also examined. A total of 25,871,325 codons were analyzed and a comprehensive codon usage table for all species was generated. This is the first codon usage table available for 24 of these organisms.

Results

Codon usage similarity in Nematoda usually persists over the breadth of a genus but then rapidly diminishes even within each clade. Globodera, Meloidogyne, Pristionchus, and Strongyloides have the most highly derived patterns of codon usage. The major factor affecting differences in codon usage between species is the coding sequence GC content, which varies in nematodes from 32% to 51%. Coding GC content (measured as GC3) also explains much of the observed variation in the effective number of codons (R = 0.70), which is a measure of codon bias, and it even accounts for differences in amino acid frequency. Codon usage is also affected by neighboring nucleotides (N1 context). Coding GC content correlates strongly with estimated noncoding genomic GC content (R = 0.92). On examining abundant clusters in five species, candidate optimal codons were identified that may be preferred in highly expressed transcripts.

Conclusion

Evolutionary models indicate that total genomic GC content, probably the product of directional mutation pressure, drives codon usage rather than the converse, a conclusion that is supported by examination of nematode genomes.

Molecular biology Evolution Genetics

Background

Utilization of the degenerate triplet code for amino acid (AA) translation is neither uniform nor random. In particular, there are distinct patterns among different species and genes. Such patterns can readily be characterized by codon usage, namely the observed percentage occurrence with which each codon is used to encode a given AA. This measure has direct utility in molecular characterization of a species in that it enables efficient degenerate and nondegenerate primer design for cross-species gene cloning, open reading frame determination, and optimal protein expression 1. Such tools are particularly important with respect to species for which limited molecular information exists. Codon usage also serves as an indicator of molecular evolution 2. Codon usage bias, namely the degree to which usage departs from uniform use of all available codons for an AA, can be influenced by a number of evolutionary processes. The guanine and cytosine (GC) versus adenine and thymine (AT) composition of the species' genome, probably the product of directional mutation pressure 34, is a key driver of both codon usage and AA composition 56. Other factors that influence codon usage may include the relative abundance of isoaccepting tRNAs 789, especially for highly expressed mRNAs that require translational efficiency 1011, presence of mRNA secondary structure 1213, and facilitation of correct co-translational protein folding 14. Codon usage appears not to be optimized to minimize the impact of errors in translation and replication 15.

Nematodes are a highly abundant and diverse group of organisms that exploit niches from free-living microbivory to plant and animal parasitism. Molecular phylogenies divide nematodes into five major named and numbered clades within which parasitism has arisen multiple times 16: Dorylaimia (clade I), Enoplia (clade II), Spirurina (clade III), Tylenchina (clade IV), and Rhabditina (clade V). Following the sequencing of the complete genome of the model nematode Caenorhabditis elegans 17, we have begun to catalog the molecular diversity of nematode genomes through the generation of over 250,000 expressed sequence tags (ESTs) from more than 30 nematode species (including 28 parasites) in four clades. Gene expression analyses for several medically and economically important parasites such as filarial, hookworm, and root knot nematode species have been completed 181920212223 (for reviews 2425). Moreover, we recently conducted a meta-analysis of partial genomes across the whole phylum with a focus on the conservation and diversification of encoded protein families 26. Project information is maintained on several online resources 27282930.

Now, in the most extensive such study yet performed for any phylum, we extend the above analyses with a comprehensive survey of observed codon usage and bias based on nearly 26 million codons in 32 species of the Nematoda. Because of its completed genome, C. elegans has been the primary species utilized in nematode codon usage studies 31323334. Our findings provide more complete information for Caenorhabditis based on all 41,782 currently predicted proteins in C. elegans and C. briggsae 35. Studies for other nematode species have been more limited. Codon usage has been tabulated for a number of parasitic nematodes including filarial species Brugia malayi, Onchocerca volvulus, Wucheria bancrofti, Acanthocheilonema viteae, Dirofilaria immitis 36373839, Strongyloides stercoralis 40, Ascaris suum 41, Ancylostoma caninum, and Necator americanus 42. Although Fadiel and coworkers 39 used up to 60 genes per species, sample sizes in the other studies were quite small, typically fewer than 10 representative genes and 5,000 codons per species. In the present study we used an average of 2,350 genes and 270,000 codons per species for the 30 non-Caenorhabditis species. Our results provide the first codon usage tables for 24 of these organisms. Web available automated codon usage databases compiled from GenBank 43 lack almost all of this information because they rely only on full-length protein coding gene sequence submissions rather than the EST data used here.

In analyzing codon distribution in Nematoda, we describe how average usage varies between species and across the phylum. For instance, it has been shown that there is a level of conservation in codon distribution between 'closely' related nematodes such as Brugia malayi and B. pahangi 37 and Brugia and Onchocerca 38. These relationships do not appear to extend over greater evolutionary distances, for instance between Onchocerca and Caenorhabditis 36. The evolutionary distance at which conservation of codon usage diminishes has not previously been established 32. Here we show that codon usage similarity in Nematoda is a relatively short-range phenomenon, generally persisting over the breadth of a genus but then rapidly diminishing within each clade. We also show that the major factor affecting differences in mean codon usage between distantly related species is the coding sequence GC as compared with AT content. GC content also explains much of the observed variation in the effective number of codons, a measure of codon bias, and even differences in AA frequency.

Results

Determination of codon usage patterns and amino acid composition

Extensive nucleotide sequence data are now available for many nematode species, largely because of recent progress using genomic approaches 2544. To obtain a better understanding of codon usage and AA composition within the phylum Nematoda, we analyzed a total of 265,494 EST sequences originating from 30 nematode species. The ESTs define 93,645 clusters or putative genes, with 208-9,511 clusters per species (Table 1) 26. Table 1 also provides two letter codes for the nematode species used throughout the remainder of the report. We used prot4EST, a translation prediction pipeline optimized for EST datasets 45, to generate protein predictions. To reduce noise derived from poor translations, our analysis considered only the longest open reading frame (ORF) translations with strong supporting evidence in the form of similarity to known or predicted proteins (BLASTX cutoff 1 × e^-8) and retained only the polypeptide aligned portion of the nucleotide sequence. About 75% of the clusters met these criteria, yielding 8,080,057 codons originating from species other than Caenorhabditis, and 25,871,325 total codons from all 32 species including available predictions from C. elegans and C. briggsae. The 18 AA residues with redundant codons gave a total of (18) × C_32,2= 496 comparisons of codon usage between species. Comprehensive tables of AA composition (Tables 2 and 3) and codon usage (Table 4) for all 32 Nematoda species studied are provided. Below we use these tables to examine, first, variation in AA composition and its relationship to GC content and, second, codon usage and its relationship to GC content.

Table 1

Summary of sequences used by nematode species

Clade

Code

Species

ESTs

Total number of clusters

Clusters or genes used

Codons

GC content (%)

Necator americanus ^a

4,766

2,294

1,784

192,756

Ancylostoma caninum ^b

9,079

4,203

3,207

305,036

Ancylostoma ceylanicum ^b

10,544

3,485

2,814

387,372

Nippostrongylus brasiliensis ^b

1,234

742

630

75,934

Haemonchus contortus ^b

17,268

4,146

4,102

584,513

Ostertagia ostertagi ^b

6,670

2,355

1,961

222,616

Teladorsagia circumcincta ^b

4,313

1,655

1,616

194,351

Caenorhabditis elegans ^c

22,254

100

9,784,215

Caenorhabditis briggsae ^c

19,528

100

8,007,053

Pristionchus pacificus ^c

8,672

3,690

2,597

297,605

IVa

Strongyloides stercoralis ^a

11,236

3,635

2,803

367,308

Strongyloides ratti ^b

9,932

3,264

2,682

320,874

Parastrongyloides trichosuri ^b

7,712

3,086

2,457

284,785

IVb

Pratylenchus penetrans ^d

1,908

408

338

45,802

Globodera pallida ^d

1,317

977

479

65,699

Globodera rostochiensis ^d

5,905

2,851

2,192

290,614

Heterodera glycines ^d

18,524

7,198

5,564

742,990

Meloidogyne incognita ^d

12,394

4,408

3,214

366,435

Meloidogyne javanica ^d

5,282

2,609

2,086

203,135

Meloidogyne arenaria ^d

3,251

1,892

1,483

176,816

Meloidogyne hapla ^d

13,462

4,479

3,507

407,985

Meloidogyne chitwoodi ^d

7,036

2,409

1,906

205,612

Zeldia punctata ^c

388

208

102

16,723

III

Ascaris suum ^b

38,944

8,482

5,830

646,740

Ascaris lumbricoides ^a

1,822

853

508

42,919

Toxocara canis ^b

4,206

1,447

866

103,065

Brugia malayi ^a

25,067

9,511

6,483

561,296

Dirofiliaria immitis ^b

3,585

1,747

1,380

126,880

Onchocerca volvulus ^a

14,922

5,097

2,914

299,336

Trichinella spiralis ^a

10,384

3,680

2,693

290,794

Trichuris muris ^b

2,713

1,577

1,179

147,995

Trichuris vulpis ^b

2,958

1,257

1,000

106,071

^aHuman parasite, ^banimal parasite, ^cfree-living, and ^dplant parasite. EST, expressed sequence tag.

Table 2

Amino acid composition (%) of translations by nematode species

Clade

Species

Amino acid

Ala

Cys

Asp

Glu

Phe

Gly

His

Ile

Lys

Leu

Met

Asn

Pro

Gln

Arg

Ser

Thr

Val

Typ

Tyr

6.9

2.4

5.2

6.2

4.7

6.3

2.5

5.5

6.5

8.6

2.6

4.2

5.0

3.7

6.2

7.2

5.3

6.5

1.2

3.3

7.0

2.4

5.0

6.0

4.7

5.9

2.7

5.6

6.3

8.9

2.8

4.2

4.6

3.7

6.2

7.4

5.4

6.6

1.3

3.3

7.6

2.2

5.5

6.6

4.2

6.5

2.5

5.2

6.4

8.5

2.5

4.0

5.1

3.8

6.1

7.1

5.3

6.7

1.2

3.0

7.8

2.2

5.4

6.3

4.0

6.9

2.4

5.0

7.2

8.1

2.6

4.0

4.9

3.5

6.4

7.0

5.2

6.9

1.1

3.1

7.4

2.3

5.5

6.5

4.3

6.6

2.5

5.4

7.0

8.4

2.5

4.1

4.9

3.7

6.0

6.4

5.2

6.8

1.2

3.4

7.2

2.3

5.3

6.3

4.4

6.7

2.6

5.3

6.7

8.4

2.6

4.0

5.2

3.8

6.1

6.8

5.3

6.7

1.1

3.1

7.5

2.7

5.2

6.1

4.3

6.7

2.6

5.0

6.6

8.4

2.7

4.2

5.1

3.8

5.8

7.1

5.3

6.5

1.2

3.3

6.3

2.0

5.3

6.5

4.8

5.4

2.3

6.1

6.4

8.6

2.6

4.9

4.2

5.2

8.1

5.9

6.2

1.1

3.1

6.3

2.0

5.3

6.8

4.7

5.4

2.3

6.0

6.4

8.5

2.6

4.8

5.0

4.2

5.4

8.0

5.8

6.1

1.1

3.1

7.4

1.9

5.4

6.9

4.0

6.6

2.5

5.3

6.6

8.4

2.6

3.9

5.1

3.4

6.4

7.6

5.4

6.4

1.2

3.0

IVa

5.5

1.9

5.7

7.0

4.3

6.0

2.1

7.1

8.0

8.3

2.3

6.0

4.5

3.7

4.6

7.2

5.5

5.8

1.0

3.5

5.4

2.0

5.4

6.5

4.7

5.9

2.1

7.4

8.1

8.6

2.4

6.2

4.3

3.6

4.3

7.2

5.4

5.8

1.0

3.8

6.3

2.0

5.3

6.3

4.6

6.3

2.4

6.6

8.1

8.3

2.4

5.3

4.3

3.5

4.9

6.9

5.5

6.1

1.0

3.7

IVb

6.9

2.0

5.3

6.7

4.3

7.0

2.4

5.8

7.6

8.4

2.6

4.5

6.2

6.4

4.9

5.9

1.2

2.9

6.8

2.4

4.5

5.4

5.6

7.2

2.4

4.9

7.1

9.1

2.3

3.9

5.8

3.8

6.7

7.1

4.8

6.0

1.2

2.9

7.4

2.1

4.9

6.0

4.8

6.5

2.6

5.2

5.9

9.3

2.5

4.3

5.0

4.4

6.5

7.2

5.2

6.3

1.2

2.7

7.3

2.2

4.9

6.2

5.1

6.3

2.6

5.2

6.0

9.2

2.4

4.5

5.0

4.5

6.3

7.3

5.1

6.2

1.2

2.5

5.7

2.0

4.8

6.7

5.1

5.6

2.2

6.5

7.3

9.4

2.3

5.6

4.5

4.6

5.3

7.5

5.2

5.4

1.1

3.0

5.5

2.1

4.8

6.6

5.5

5.4

2.2

6.9

7.8

9.6

2.4

5.6

4.2

5.4

7.0

5.1

5.4

1.1

3.3

5.7

2.0

5.0

6.9

5.3

5.7

2.2

6.9

7.3

9.7

2.3

5.5

4.1

4.3

5.2

7.0

5.0

5.6

1.1

3.2

5.6

2.0

4.9

6.8

5.3

5.6

2.2

7.0

7.3

9.5

2.3

5.8

4.3

5.2

7.3

4.9

5.4

1.2

3.2

5.3

2.3

4.8

6.4

5.5

2.3

7.4

9.7

2.3

6.0

4.0

4.3

4.9

7.4

5.0

5.2

1.1

3.3

7.6

1.5

5.2

6.2

4.2

7.1

2.5

6.1

7.9

8.4

1.8

4.7

4.6

3.8

6.0

5.4

5.5

6.6

1.1

3.7

III

7.0

2.5

4.9

6.1

4.6

5.8

2.5

6.0

6.3

8.7

2.6

4.5

4.8

3.6

6.3

7.5

5.3

6.6

1.2

3.3

7.3

2.6

4.6

5.8

4.9

6.1

2.5

6.0

6.4

8.3

2.5

4.3

5.3

3.5

6.2

7.4

5.2

6.5

1.2

3.4

7.3

2.8

5.0

6.0

4.2

6.8

2.6

5.3

7.3

8.0

2.4

4.1

5.4

3.5

6.2

6.7

5.6

6.4

1.2

3.2

5.6

2.5

4.6

5.6

5.4

5.0

2.6

7.1

6.8

9.6

2.8

5.0

4.1

3.8

5.6

7.8

5.3

5.9

1.1

3.6

5.6

2.4

4.9

6.0

5.2

4.7

2.7

7.5

7.0

9.5

2.7

5.2

3.9

3.8

5.9

7.5

5.1

5.8

1.1

3.8

6.0

2.2

5.0

6.1

4.9

5.6

2.5

6.9

7.1

8.9

2.7

4.9

4.5

3.9

5.9

7.3

5.2

5.7

1.2

3.5

6.2

2.6

5.0

6.2

5.1

2.5

6.1

6.6

9.5

2.6

4.9

4.1

3.9

5.6

7.6

5.1

6.5

1.2

3.4

7.1

3.0

5.0

6.0

4.5

6.5

2.5

5.0

6.3

8.9

2.6

4.0

5.0

3.8