A random sequencing approach for the analysis of the Trypanosoma cruzi genome: general structure, large gene and repetitive DNA families, and gene discovery

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A random sequencing approach for the analysis of the Trypanosoma cruzi genome: general structure, large gene and repetitive DNA families, and gene discovery
  A Random Sequencing Approach for the Analysisof the  Trypanosoma cruzi   Genome: General Structure,Large Gene and Repetitive DNA Families, andGene Discovery Ferna´n Agu¨ero, 1 Ramiro E. Verdu´n, 1  Alberto Carlos C. Frasch,and Daniel O. Sa´nchez 2 Instituto de Investigaciones Biotecnolo´gicas, Instituto Tecnolo´gico de Chascomu´s, Universidad Nacional de General SanMartı ´n, Consejo Nacional de Investigaciones Cientı ´ficas y Te´cnicas, San Martı ´n, Provincia de Buenos Aires, 1650 Argentina  A random sequence survey of the genome of   Trypanosoma cruzi  , the agent of Chagas disease, was performed and11,459 genomic sequences were obtained, resulting in  ∼ 4.3 Mb of readable sequences or  ∼ 10% of the parasitehaploid genome. The estimated total GC content was 50.9%, with a high representation of A and T di- andtrinucleotide repeats. Out of the estimated 5000 parasite genes, 947 putative new genes were identified.Another 1723 sequences corresponded to genes detected previously in  T. cruzi   through expression sequence taganalysis. 7735 sequences had no matches in the database, but the presence of open reading frames that passedFickett’s test suggests that some might contain coding DNA. The survey was highly redundant, with  ∼ 35% of the sequences included in a few large sequence families. Some of them code for protein families present indozens of copies, including proteins essential for parasite survival and retrotransposons. Other sequence familiesinclude repetitive DNA present in thousands of copies per haploid genome. Some families in the latter groupare new, parasite-specific, repetitive DNAs. These results suggest that  T. cruzi   could constitute an interestingmodel to analyze gene and genome evolution due to its plasticity in terms of sequence amplification anddivergence. Additional information can be found at http://www.iib.unsam.edu.ar/tcruzi.gss.html.[The sequence data described in this paper have been submitted to the dbGSS database under the followingGenBank accession nos.: AQ443439–AQ443513, AQ443743–AQ445667, AQ902981–AQ911366, AZ049857–AZ051184,and AZ302116–AZ302563.] Protozoan parasites from the order Kinetoplastida arethe causative agents of widespread diseases in humansas well as of considerable economic loss through infec-tionofdomesticanimalsandwildlife.Amongthemare Trypanosoma cruzi  and  Trypanosoma brucei , the agentsofChagasdiseaseintheAmericasandsleepingsicknessin Africa, respectively, and  Leishmania  spp., causing avariety of pathologies in humans. In addition to theirmedical importance, these parasites were the source of discoveries of fundamental cellular and molecular phe-nomenasuchasRNAediting(Stuartetal.1997;EstevezandSimpson1999),mRNAtrans-splicing(Nilsen1995;Lee and Van der Ploeg 1997), glycosylphosphatidyl–inositol anchoring of proteins (Krakow et al. 1986),and antigenic variation (Rudenko et al. 1998), amongothers. Kinetoplastids are also interesting evolution-arily because they represent one of the earliest eukary-otic organisms that diverged from the ancestor of themain eukaryotic branch.Initial studies on the genome structure of theseorganisms were hampered by the difficulty of applyingclassical genetic tools. As is the case with yeast, chro-mosomes do not condense during mitosis and cannotbe visualized directly with color stains. In spite of thesedrawbacks several recent studies have revealed a highlyplastic genome with an unusual gene organization(Zingalesetal.1997;Network1998;Ersfeldetal.1999).In the case of   T. cruzi  it has been shown that there is alarge chromosomal size variation between strains thatcan also be observed between pairs of homologouschromosomes (Henriksson et al. 1995).Many genes in kinetoplastids, including house-keeping genes, are present in multiple copies, eitherclustered in tandems or distributed in different chro-mosomes (Campetella et al. 1992b; El-Sayed andDonelson 1997). Recently, the complete sequence of chromosome 1 from  L. major   Friedlin (Myler et al.1999) and a partial sequence of chromosome 3 from  T.cruzi  (Andersson et al. 1998) were obtained, showing inboth cases a similar organization with two clusters of  1 These authors contributed equally to this work. 2 Corresponding author.E-MAIL dsanchez@iib.unsam.edu.ar; FAX 54-11-4752-9639.  Article published online before print:  Genome Res.,  10.1101/gr.146300. Article and publication are at www.genome.org/cgi/doi/10.1101/gr.146300. Letter 1996 Genome Research  10:1996–2005 ©2000 by Cold Spring Harbor Laboratory Press ISSN 1088-9051/00 $5.00; www.genome.org www.genome.org  genes per chromosome transcribed in opposite direc-tions. These studies also showed that the gene densityis high in the regions where genes are clustered, with ∼ 1 gene every 3.6 Kb in both cases. These and otherunusual characteristics in genome structure and orga-nization make these parasites an interesting field of study to further understand eukaryote gene and ge-nome evolution.As part of the parasite genome projects launchedby the Tropical Disease Program of the World HealthOrganization, we and others have performed expressedsequence tag (EST) analysis of the  T. cruzi  genome as ameans for rapid gene finding (Verdu ´ n et al. 1998; Por-cel et al. 2000). As a first step toward obtaining thecomplete sequence of the clone selected for the  T. cruzi genomeproject(CL-Brenerclone)wehavenowmadearandom genome survey of   ≈ 10% (4.3 Mb) of the hap-loid genome of the parasite. The results obtained al-lowed us to make a general characterization of the  T.cruzi  genome, identify putative new genes, and definelarge gene families and repetitive sequence familiespresent in the parasite genome, including a novel re-petitive element and uncharacterized abundant se-quences. RESULTS AND DISCUSSION Overall Structure of the  T. cruzi   Genome A random genomic library of   T. cruzi  DNA was con-structed and used to produce 11,459 reads with an av-erage length of 374 bp after vector removal and qualityclipping. These GSS (Genomic Sequence Survey) se-quences represent 4.3 Mb of readable sequences or ∼ 10% of the parasite haploid genome, which is  ∼ 40 Mb(Frohme et al. 1998). The total GC content of the se-quence produced was 50.9%, a value that is slightlylarger than the one obtained from estimations madeon 8796  T. cruzi  ESTs (49.6 %), and the one obtainedfor 93.4 Kb from chromosome 3 (48.5%) (Andersson etal. 1998). The fractional GC content for all the GSSsvaried from 0.18 – 0.71, with  ∼ 69% of the sequencesfalling in the 0.47 – 0.57 range.The frequency of di- and trinucleotide repeats cal-culatedforalltheGSSsequences(11,459GSS)isshownin Figure 1.  T. cruzi  has levels of CpG, TpG, and CpAdinucleotides which are near their expected values, un-like mammalian or vertebrate genomes that show asuppressionoftheCpGdinucleotideandincreasedlev-els of the TpG and CpA dinucleotides (Regev et al.1998). The TpA dinucleotide, however, is suppressed in T. cruzi , with an observed/expected ratio of 0.54. Thisdinucleotide is also suppressed in mammalian ge-nomes (Smith and Waterman 1983), and in  Fugu  thepufferfish (Elgar et al. 1999), although the reason forthis is not known. Also, the abundance of (A)n and(T)n di- and trinucleotides is remarkable. The fre-quency of these di- and trinucleotides is greater thanthe observed frequency for the CpA dinucleotide, themost common dinucleotide in vertebrates. When thesame analysis was done for the partial sequence of chromosome 3, we obtained similar values, except forthe (A)n and (T)n di- and tri-nucleotides. These alsorepresent the most frequent species, but their ob-served/expected ratios are higher. In our survey the(A)ndinucleotidehada0.13obs/expratio,whereasthepartial sequence of chromosome 3 had a 0.4 ratio. Forthe (A)n trinucleotide these values are higher: a 0.67ratio in our survey and a 1.43 ratio in the partial se-quence of chromosome 3. Coding Content of the GSS Sequences Wecomparedthesequencesinourdatasettoaprotein Figure 1  Frequency of di- and tri-nucleotide repeats in the  Try-panosoma cruzi   genome. The total of 11,459 sequences wereused to search for the occurrence of all possible words of length2 ( A ) and 3 ( B  ) on both strands of the sequences using  COMPSEQ .Theexpectedfrequencyofeachwordisbasedontheassumptionthat all words have the same probability of occurrence. Di- andtri-nucleotide frequencies are expressed as Observed (Obs)/Expected (Exp)  1, so that negative values correspond to sup-pressed di- and tri-nucleotides and positive values correspond todi- and tri-nucleotides with frequencies over that expected. Be-cause the search is done on both strands, only one reversecomplementary di- or tri-nucleotide of a pair is shown. Trypanosoma cruzi   Random Genomic Sequencing Genome Research 1997 www.genome.org  nonredundant database using  BLASTX  (Altschul et al.1997). From this analysis, 3724 GSS (32.5%) showedsignificant (E<1e-5) similarities to sequences present inthe public database (GenBank release 115) (Table 1);2778 GSS (24.2%) matched  T. cruzi  sequences while947GSS(8.3%)matchednon- T. cruzi sequences.67.5%did not match any sequence in the database. When wecompared each GSS to the dbEST database, only 1723(15%) of the GSSs gave significant matches and 96% of these were matches to  T. cruzi  ESTs. The low hit rateagainst ESTs could be explained in part by the low se-quence coverage attained in the EST sequencing,which only covered one of the four main life cyclestages of the parasite. Thus, the coding content of oursurvey could be considered to be 15% based on thenumber of matches against dbEST or 32.5% based onthe number of matches against proteins in nonredun-dant databases. However, this does not include se-quences absent from the databases.To detect putative coding sequences using a differ-ent criteria, we first searched our sequences for openreading frames (ORFs) > 300 bp (defined as sequenceswithout a stop codon and no requirement for startcodons). We then evaluated which of these resultingsequences could be regarded as coding using the test-code algorithm developed by Fickett (1982), whichmeasures the positional randomness of a sequence,and is independent of the reading frame. About 83% of the sequences (9520 GSSs) had ORFs with the men-tioned requirements, and 23% of these (2222 GSSs)were found to be potentially coding by the testcodealgorithm. Using this figure as the minimum numberof coding sequences in our survey, we can estimate thenumber of genes for  T. cruzi  to be about 5000 per hap-loid genome (considering an average gene size of 1500bp and a haploid genome size of 40 Mbp). Identification of Putative New Genes Based on the  BLASTX  analysis, which is summarized inTable 1, we detected 947 putative new genes, which arethosepositivematchesagainstanon- T. cruzi proteininthe nonredundant database. The best 50 matches areshown in Table 2 (detailed information on  T. cruzi  GSSsequences can be found at http://www.iib.unsam-.edu.ar/tcruzi/gss.html).Among the new genes found there is a GSS(GSSTc12036) with similarity to N-myristoyl transfer-ases (Nmt). Nmt is only found in eukaryotic cells andtransfers fatty acid myristate from myristoyl-CoA tothe amino-terminal glycine of substrate proteins (Rus-sell Johnson et al. 1994). Genetic and biochemicalstudies have established Nmt as a target for the devel-opment of a new class of fungicidal drugs, and thestructure of Nmt from two lower eukaryotes, namely Saccharomyces  and  Candida  has been solved (Weston etal. 1998; Bhatnagar et al. 1999). In trypanosomes pro-tein N-myristoylation has not yet been demonstrated,whereas it has been shown that these parasites can doS-myristoylation of proteins (Armah and Mensa-Wilmot1999).OtherinterestingfindingsweretwoGSShomologous to proteins involved in chromatin remod-eling. GSSTc788 is homologous to the  Drosophila  ISWIprotein, which is part of several ATP-dependent chro-matin remodeling complexes such as NURF (Nucleo-some remodeling factor), CHRAC (chromatin accessi-bility complex) and ACF (ATP-utilizing chromatin as-sembly and remodeling factor) (Muchardt and Yaniv1999), whereas GSSTc11568 is homologous to severalhistone deacetylases. Chromatin remodeling is amechanism of transcriptional regulation that has beendemonstrated in many eukaryotes including yeast,which as trypanosomes does not show chromosomecondensation during its cell cycle. Another GSS(GSSTc12012) had homology with a  T. brucei  VSG ex-pression site-associated protein precursor (ESAG-2),which is a member of a large gene family that includesnonfunctional genes (Kooter et al. 1988). Also, severalGSSs with homology to proteins having RNA bindingdomains were identified, including one clone(GSSTc11533)thatshowedhomologytotheRNAbind-ing domain present in the developmentally regulatedproteinsp37andp34from T. brucei (Zhangetal.1998). Identification of Large Gene Families To identify large gene families, GSS sequences wereclustered using the  PHRAP  program to assemble con-tigs. Using this method we were able to group 7883reads in 2091 contigs; the other 3576 were singlets(reads having no nonvector match to any other read).This means that our survey contains 5667 unique se-quences, according to our clustering method, and thusa redundancy of at least  ∼ 50%. Further clustering ispossible, however, because the results from  BLASTX contained several GSS that belong to different contigsshowing matches to the same sequences in the data-base. To estimate the total number of GSS that belong Table 1.  BLASTX Matches to Nonredundant DatabasesNo.of GSS% of GSSNo.ESTs% of ESTs Total 11,459 100 8,796 100Database matches:Total 3,724 32.5 2,552 29 T. cruzi   2,778 24.2 861 9.8Other trypanosomatids 331 2.9 262 3Other organisms 616 5.4 1,429 16.2No database match 7,735 67.5 6,244 71 All of the GSSs presented in this paper and all the  T. cruzi   ESTspresent in the dbEST division of GenBank were used to searchthe NCBI nonredundant database (GenBank Release 115).Matches with an  E   value  1e-5 were considered positive. Agu ¨ ero et al. 1998 Genome Research www.genome.org  to a given gene family, we used the consensus se-quence from each contig to search a local  T. cruzi  GSSdatabase using  BLASTN . Based on this analysis we wereable to delineate abundant sequences in our survey,which are the main contributors to the observed re-dundancy. The most abundant gene families in the  T.cruzi  genome are summarized in Table 3.Among the largest families identified is the super-family of   T. cruzi  antigens, also known as trans-sialidase-like molecules (632 copies per haploid ge-nome). Their members have a number of different ac-tivities, most of them involved in the host – parasiteinteraction (Frasch 2000). Other sequences alreadyknown to conform large gene families in  T. cruzi  iden- Table 2.  Identification of New  T. cruzi   GenesdbGSS a Description b Score Expect 11462 ref-NP_011059.1-GLC7-protein phosphatase type I [ Saccharomyces cereviseae  ] 474 0.00E+0010993 sp-P22679-elongation factor TU (EF-TU) [ Mycoplasma hominis  ] 312 0.00E+0000184 sp-P46794-cystathionine beta-synthase [ Dictyostelium discoldeum]   303 0.00E+0011438 sp-O76767-lumen protein retaining receptor [ Drosophila melanogaster  ] 208 0.00E+0011472 emb-CAB56598.1-alpha dynein heavy chain [ Chlamydomonas reinhardtii  ] 202 0.00E+0011026 gi-2425121-Spalten [ Dictyostelium discoldeum ] 116 0.00E+0012036 gb-AAF19802.1-N-myristoyl transferase [ Brassica oleracea  ] 107 0.00E+0001761 gl-3004644-trypanothione synthetase [ Crithidia fasciculata  ] 428 5.00E-4211137 gb-AAB67249.1-T-complex protein 1, Beta subunit [ Homo sapiens  ] 424 9.00E-4211193 ref-NP_014850.1-RET1-second-largest subunit of RNA polymerase III [ Saccharomyces cereviseae  ] 422 1.00E-4111590 gb-AAF08387.1-26S proteasome regulatory complex subunit p48A [ Drosophila melanogaster  ] 419 2.00E-4111120 emb-CAA65384-malate dehydrogenase [ Mesembryanthemum crystallinum ] 371 2.00E-3511285 gi-1931649-DNA helicaso isolog [ Arabidopsis thaliana  ] 353 2.00E-3311563 ref-NP_013458.1-transaldolase [ Saccharomyces cerevisiae  ] 348 7.00E-3309938 gi-2246458-S-adenosyl-methionine-sterol-C-methyltransferase [ Ricinus communis  ] 348 8.00E-330705 pir-T1017324-sterol C-methyltransferase-castor bean [ Ricinus communis  ] 348 9.00E-3311338 gb-AAC02737.1-3-hydroxyisobutyryl-coenzyme A hydrolase [ Arabidopsis thaliana  ] 341 5.00E-3211467 gb-AAF04493.1-acetyl-CoA carboxylase 1 [ Toxoplasma gondii]   330 7.00E-3110956 dbj-BAA84364.1-DEIH-box RNA/DNA helicase [ Arabidopsis thaliana  ] 328 2.00E-3011575 gb-AAC73040.1-putative AAA-type ATPase [ Arabidopsis thaliana  ] 323 5.00E-3011516 sp-P05439-ATP synthase alpha chain [ Rhodobacter blasticus  ] 319 2.00E-2911606 sp-P51044-citrate synthase, mitochondrial precursor [ Aspergillus niger  ] 321 2.00E-2811417 gb-AAF21464.1-proline oxidase 2 [ Homo sapiens  ] 310 2.00E-2811502 gi-2654103-MAPKK kinase [ Neurospora crassa  ] 304 9.00E-2811523 gb-AAD26855.1-phenylalanyl tRNA synthetase beta subunit [ Mus musculus  ] 301 2.00E-2711568 gi-4101722-histone deacetylase mHDA1 [ Mus musculus  ] 301 2.00E-2711480 gi-2462752-phosphatidylinositol 3-kinase [ Arabidopsis thaliana  ] 299 4.00E-2711463 sp-P32826-serine carboxypeptidase precursor [ Arabidopsis thaliana  ] 299 4.00E-2710965 gi-687208-dynein heavy chain isotype 5C [ Tripneustes gratilla  ] 289 5.00E-2611328 pir-A56220-protein kinase aurora-fruit fly [ Drosophila melanogaster  ] 287 1.00E-2511446 sp-O15228-dihydroxyacetone phosphate acyltransferase (DAP-AT) [ Homo sapiens  ] 280 7.00E-2511433 gb-AAF11511.1-acetyl-CoA acetyltransferase [ Deinococcus radiodurans  ] 279 9.00E-2511601 sp-P30575-enolase 1 (2-phosphoglycerate dehydratase) [ Candida albicans  ] 278 1.00E-2401810 sp-O94476-eukaryotic translation initiation factor 6 (EIF-6) [ Schizosaccharomyces pombe  ] 275 3.00E-240740 gb-AAF62506.1-ribosomal protein LS [ Trypanoplasma borreli  ] 273 5.00E-2411274 pir-S70896-aminomethyltransferase [ Saccharomyces cerevisiae  ] 271 5.00E-2411279 gi-780410-helicase [African swine fever virus] 272 6.00E-2411882 sp-Q07405-ATP synthase alpha chain [ Myxococcus xanthus  ] 269 1.00E-2311432 emb-CAB40791.1-centrin [ Euplotes octocarinatus  ] 265 3.00E-2311654 gi-1872473-delta-24-sterol methyltransferase [ Triticum aestivum ] 258 7.00E-2301015 pir-A56492-protein kinase ERK2 [ Dictyostelium discoideum ] 280 8.00E-2311584 ref-NP_005678.1-phenylalanyl-tRNA synthetase beta-subunit [ Homo sapiens  ] 259 1.00E-2211434 sp-O05593-methionyl-tma synthetase [ Mycobacterium tuberculosis  ] 254 6.00E-2211038 gi-1354084-axonemal dynein light chain p33 ( Strongylocentrotus purpuratus  ] 251 2.00E-2111440 gi-2665637-mismatch repair protein MSH6 [ Mus musculus  ] 248 4.00E-2111248 gb-AAF22155.1-ARD-1 N-acetyltransferase homologue [ Mus musculus  ] 244 1.00E-2011852 gb-AAC32590.1-sperm flagellar protein Repro-SA-1 [ Homo sapiens  ] 239 5.00E-2001825 dbj-BAA20996-kinesin-like protein [ Caenorhabditis elegans  ] 239 6.00E-2011210 pir-A35630-regulatory protein algR3 [ Pseudomonas aeruginosa  ] 237 7.00E-20GSS sequences were used to search NCBI ’ s non-redundant database using BLASTX. The first 50 GSSs out of the 947 GSSs showingmatches against non- T. cruzi   sequences are listed. Detailed information about the homologies found for GSSs can be found athttp://www.iib.unsam.edu.ar/genomelab/tcruzi/gss.html. a GSS names in dbGSS are the numbers given here preceded by GSSTc (e.g., GSSTc11210). b Descriptions are taken directly from the BLAST reports. Trypanosoma cruzi   Random Genomic Sequencing Genome Research 1999 www.genome.org  tified through this screening were the cysteine protein-ase cruzipain (Campetella et al. 1992a), dgf-1, a largeprotein of unknown function (Wincker et al. 1992),and the parasite mucins (Di Noia et al. 1998). In all of these cases, the estimated number of copies agreed wellwith experimental data, showing that our sampling Table 3.  Large Gene Families in  T. cruzi  A Gene Families  No. of GSSsEstimated No.of CopiesRelative%No. of ESTs Reference dgf-1 494 154 4.3 6 (Wincker et al. 1992)trans-sialidase 427 632 3.7 10 (Parodi et al. 1992)L1 non-LTR retrotransposon 214 149 1.9 5 (Mart í  n et al. 1995)Mucin 122 710 1.1 5 (Di Noia et al. 1995)Cysteine proteinase (Cruzipain) 39 91 0.3 7 (Campetella et al. 1992a)predicted ORF (gi3053534), chromosome 3 38 103 0.3 4 (Andersson et al. 1998)gp63 34 70 0.3 9 AF110951, unpubl.Histone H4 29 337 0.2 10 (Soto et al. 1997)Casein kinase homolog 23 81 0.2 7 AF089709, unpubl. Adenylyl cyclase 19 18 0.2 0 (Taylor et al. 1999)Hsp70 18 25 0.2 48 (Requena et al. 1988)Histone H2A 17 145 0.1 106 (Puerta et al. 1994)Helicase 14 24 0.1 15Hsp90 11 18 0.1 7 (Mottram et al. 1989)Total 1499 14.5 B Repetitive DNA Families  No. of GSSsEstimated No.of CopiesRelative%No. of ESTs Reference minichromosomal 195 bp repeat 854 15287 7.45 ND (Gonzalez et al. 1984)TcIRE (I) 266 1664 2.3 8 this work VIPER 174 257 1.5 ND (V  á zquez et al. 2000)C6 interspersed element 230 560 2.0 ND (Araya et al. 1997)SIRE 201 3011 1.8 ND (V  á zquez et al. 2000)telomere associated sequences 131 1963 1.1 NDTcIRE (II) 47 2310 0.4 2 this workTRBSEQA 31 105 0.3 ND (Requena et al. 1992)HCR6 10 57 0.1 ND (de Mendon ç a-Lima and Traub-Cseko 1991)Spliced Leader gene 12 69 0.1 NDTotal 2133 18.6 C Unknown Families  No. of GSSsEstimated No.of CopiesRelative%No. of ESTs Consensus Size (bp) Cluster 2009 19 54 0.1 0 1220Cluster 2047 85 136 0.7 1 2170Cluster 2015 53 96 0.5 0 1917Cluster 1994 25 82 0.2 1 1056Cluster 2056 22 30 0.2 0 2571Cluster 2019 21 102 0.2 3 1051Cluster 2027 12 58 0.1 0 718Cluster 1986 10 48 0.1 0 728Total 247 2.1GSS sequences were clustered and their similarities against sequences in nonredundant databases were determined. Total number of GSS for each family was determined as described in the text. Note, however, that this approach can lead to a GSS belonging to morethan one family. To calculate the number of copies, the value of the gene size (GS) used was the length of the coding sequence(excluding UTRs) of a representative member from each family; in the case of genes with different sizes, an average was used. Whenonly partial sequences were available, copy numbers were not determined as this could lead to overestimation of the figures. Todetermine the number of ESTs for a given gene family, the consensus sequence or a sequence of a representative member was usedto do a BLASTN search against the 8796  T. cruzi   ESTs. Matches with E < 1e-40 were considered positive. In the case of unpublishedsequences that are available from nucleotide databases, the GenBank accession number is given. (A) Gene families (protein coding).(B) Repetitive DNA families (likely to be noncoding). (C) Uncharacterized sequences described in this work. Information about theseunknown families (consensus sequence and individual GSSs included in the contig) can be found at http://www.iib.unsam.edu.ar/genomelab/tcruzi/gss.html. Agu ¨ ero et al. 2000 Genome Research www.genome.org
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