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Chromosome level assembly of the hybrid Trypanosoma cruzi genome.

Weatherly DB, Boehlke C, Tarleton RL - BMC Genomics (2009)

Bottom Line: The approach was substantiated through the use of Southern blot analysis to confirm the mapping of BAC clones using as probes the genes they are predicted to contain, and each chromosome construction was visually validated to ensure sufficient evidence was present to support the organization.Now assembled, these chromosomes bring T. cruzi to the same level of organization as its kinetoplastid relatives and have been used as the basis for the T. cruzi genome in TriTrypDB, a trypanosome database of EuPathDB.In addition, they will provide the foundation for analyses such as reverse genetics, where the location of genes and their alleles and/or paralogues is necessary and comparative genome hybridization analyses (CGH), where a chromosome-level view of the genome is ideal.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA, USA. dbrentw@uga.edu

ABSTRACT

Background: In contrast to the essentially fully assembled genome sequences of the kinetoplastid pathogens Leishmania major and Trypanosoma brucei the assembly of the Trypanosoma cruzi genome has been hindered by its repetitive nature and the fact that the reference strain (CL Brener) is a hybrid of two distinct lineages. In this work, the majority of the contigs and scaffolds were assembled into pairs of homologous chromosomes based on predicted parental haplotype, inference from TriTryp synteny maps and the use of end sequences from T. cruzi BAC libraries.

Results: Ultimately, 41 pairs of chromosomes were assembled using this approach, a number in agreement with the predicted number of T. cruzi chromosomes based upon pulse field gel analysis, with over 90% (21133 of 23216) of the genes annotated in the genome represented. The approach was substantiated through the use of Southern blot analysis to confirm the mapping of BAC clones using as probes the genes they are predicted to contain, and each chromosome construction was visually validated to ensure sufficient evidence was present to support the organization. While many members of large gene families are incorporated into the chromosome assemblies, the majority of genes excluded from the chromosomes belong to gene families, as these genes are frequently impossible to accurately position.

Conclusion: Now assembled, these chromosomes bring T. cruzi to the same level of organization as its kinetoplastid relatives and have been used as the basis for the T. cruzi genome in TriTrypDB, a trypanosome database of EuPathDB. In addition, they will provide the foundation for analyses such as reverse genetics, where the location of genes and their alleles and/or paralogues is necessary and comparative genome hybridization analyses (CGH), where a chromosome-level view of the genome is ideal.

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Validation of assemblies by Southern "dot" blot analysis. a) The top-most cartoon depicts overlapping BAC clones (light blue) which are predicted to span a chromosome (assembled from multiple scaffolds, dark blue). Genes (orange) are selected such that they link proposed contiguous scaffolds. These overlapping pairs of probes create a stair-stepping effect on the Southern blot if the hybridization results are as predicted (bottom), indicating that the linkage of the scaffolds, and thus the chromosome, is correct. b) TcChr20 was chosen for Southern blot validation. Overlapping BAC clones (grey) are selected such that they span most of the chromosome. Gene probes are shown in orange (ids shown are truncated forms of the "Tc" gene ids, i.e. 506581.10 denotes Tc00.1047053506851.10). While there are 11 previously published scaffolds linked on this chromosome, the red stars indicate a region containing many trans-sialidase genes whose repetitive nature must have hindered the joining of the 4 scaffolds that terminate in this region (2 large "P" scaffolds and 2 "S" scaffolds). c) For each BAC clone, both of the gene probes positively hybridize as predicted, creating the stair-step effect that confirms the linkage of the genes on the chromosome. BAC clones are labeled with their assigned ids from the CHORI library.
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Figure 3: Validation of assemblies by Southern "dot" blot analysis. a) The top-most cartoon depicts overlapping BAC clones (light blue) which are predicted to span a chromosome (assembled from multiple scaffolds, dark blue). Genes (orange) are selected such that they link proposed contiguous scaffolds. These overlapping pairs of probes create a stair-stepping effect on the Southern blot if the hybridization results are as predicted (bottom), indicating that the linkage of the scaffolds, and thus the chromosome, is correct. b) TcChr20 was chosen for Southern blot validation. Overlapping BAC clones (grey) are selected such that they span most of the chromosome. Gene probes are shown in orange (ids shown are truncated forms of the "Tc" gene ids, i.e. 506581.10 denotes Tc00.1047053506851.10). While there are 11 previously published scaffolds linked on this chromosome, the red stars indicate a region containing many trans-sialidase genes whose repetitive nature must have hindered the joining of the 4 scaffolds that terminate in this region (2 large "P" scaffolds and 2 "S" scaffolds). c) For each BAC clone, both of the gene probes positively hybridize as predicted, creating the stair-step effect that confirms the linkage of the genes on the chromosome. BAC clones are labeled with their assigned ids from the CHORI library.

Mentions: We initially sought to validate the linkage of genes on the same chromosomes using data from previously published physical maps [16-18,20-22,27]. However, this effort was ultimately inconclusive, as too few of the described markers mapped unambiguously to single chromosomes. Rather than attempt further chromosomal blot analyses, we chose to validate the method of chromosome assembly by probing mapped, overlapping BAC clones with specific genes that the clones were predicted to contain based upon our assembly (Figure 3a, b). Figure 3c shows that in all cases, the probes hybridized with the expected BAC clones. These results substantiate the utility of our methods, as the linkage of 2 large scaffolds on the non-Esmeraldo-like "P" chromosome and 5 scaffolds on the Esmeraldo-like "S" chromosome was confirmed. The gap in the middle of the chromosome between scaffolds CH473364 and CH473349 on the P chromosome and CH473583 and CH473541 on the S chromosome (denoted by red stars on Figure 3b) shows a region rich in trans-sialidase sequences that may have hindered the linkage of these scaffolds in the original assembly. Although the complete chromosomal assembly was not validated by dot blot analysis, similar probing of other selected chromosome regions also confirmed the quality of the assembly (Additional File 1 and text below).


Chromosome level assembly of the hybrid Trypanosoma cruzi genome.

Weatherly DB, Boehlke C, Tarleton RL - BMC Genomics (2009)

Validation of assemblies by Southern "dot" blot analysis. a) The top-most cartoon depicts overlapping BAC clones (light blue) which are predicted to span a chromosome (assembled from multiple scaffolds, dark blue). Genes (orange) are selected such that they link proposed contiguous scaffolds. These overlapping pairs of probes create a stair-stepping effect on the Southern blot if the hybridization results are as predicted (bottom), indicating that the linkage of the scaffolds, and thus the chromosome, is correct. b) TcChr20 was chosen for Southern blot validation. Overlapping BAC clones (grey) are selected such that they span most of the chromosome. Gene probes are shown in orange (ids shown are truncated forms of the "Tc" gene ids, i.e. 506581.10 denotes Tc00.1047053506851.10). While there are 11 previously published scaffolds linked on this chromosome, the red stars indicate a region containing many trans-sialidase genes whose repetitive nature must have hindered the joining of the 4 scaffolds that terminate in this region (2 large "P" scaffolds and 2 "S" scaffolds). c) For each BAC clone, both of the gene probes positively hybridize as predicted, creating the stair-step effect that confirms the linkage of the genes on the chromosome. BAC clones are labeled with their assigned ids from the CHORI library.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2698008&req=5

Figure 3: Validation of assemblies by Southern "dot" blot analysis. a) The top-most cartoon depicts overlapping BAC clones (light blue) which are predicted to span a chromosome (assembled from multiple scaffolds, dark blue). Genes (orange) are selected such that they link proposed contiguous scaffolds. These overlapping pairs of probes create a stair-stepping effect on the Southern blot if the hybridization results are as predicted (bottom), indicating that the linkage of the scaffolds, and thus the chromosome, is correct. b) TcChr20 was chosen for Southern blot validation. Overlapping BAC clones (grey) are selected such that they span most of the chromosome. Gene probes are shown in orange (ids shown are truncated forms of the "Tc" gene ids, i.e. 506581.10 denotes Tc00.1047053506851.10). While there are 11 previously published scaffolds linked on this chromosome, the red stars indicate a region containing many trans-sialidase genes whose repetitive nature must have hindered the joining of the 4 scaffolds that terminate in this region (2 large "P" scaffolds and 2 "S" scaffolds). c) For each BAC clone, both of the gene probes positively hybridize as predicted, creating the stair-step effect that confirms the linkage of the genes on the chromosome. BAC clones are labeled with their assigned ids from the CHORI library.
Mentions: We initially sought to validate the linkage of genes on the same chromosomes using data from previously published physical maps [16-18,20-22,27]. However, this effort was ultimately inconclusive, as too few of the described markers mapped unambiguously to single chromosomes. Rather than attempt further chromosomal blot analyses, we chose to validate the method of chromosome assembly by probing mapped, overlapping BAC clones with specific genes that the clones were predicted to contain based upon our assembly (Figure 3a, b). Figure 3c shows that in all cases, the probes hybridized with the expected BAC clones. These results substantiate the utility of our methods, as the linkage of 2 large scaffolds on the non-Esmeraldo-like "P" chromosome and 5 scaffolds on the Esmeraldo-like "S" chromosome was confirmed. The gap in the middle of the chromosome between scaffolds CH473364 and CH473349 on the P chromosome and CH473583 and CH473541 on the S chromosome (denoted by red stars on Figure 3b) shows a region rich in trans-sialidase sequences that may have hindered the linkage of these scaffolds in the original assembly. Although the complete chromosomal assembly was not validated by dot blot analysis, similar probing of other selected chromosome regions also confirmed the quality of the assembly (Additional File 1 and text below).

Bottom Line: The approach was substantiated through the use of Southern blot analysis to confirm the mapping of BAC clones using as probes the genes they are predicted to contain, and each chromosome construction was visually validated to ensure sufficient evidence was present to support the organization.Now assembled, these chromosomes bring T. cruzi to the same level of organization as its kinetoplastid relatives and have been used as the basis for the T. cruzi genome in TriTrypDB, a trypanosome database of EuPathDB.In addition, they will provide the foundation for analyses such as reverse genetics, where the location of genes and their alleles and/or paralogues is necessary and comparative genome hybridization analyses (CGH), where a chromosome-level view of the genome is ideal.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA, USA. dbrentw@uga.edu

ABSTRACT

Background: In contrast to the essentially fully assembled genome sequences of the kinetoplastid pathogens Leishmania major and Trypanosoma brucei the assembly of the Trypanosoma cruzi genome has been hindered by its repetitive nature and the fact that the reference strain (CL Brener) is a hybrid of two distinct lineages. In this work, the majority of the contigs and scaffolds were assembled into pairs of homologous chromosomes based on predicted parental haplotype, inference from TriTryp synteny maps and the use of end sequences from T. cruzi BAC libraries.

Results: Ultimately, 41 pairs of chromosomes were assembled using this approach, a number in agreement with the predicted number of T. cruzi chromosomes based upon pulse field gel analysis, with over 90% (21133 of 23216) of the genes annotated in the genome represented. The approach was substantiated through the use of Southern blot analysis to confirm the mapping of BAC clones using as probes the genes they are predicted to contain, and each chromosome construction was visually validated to ensure sufficient evidence was present to support the organization. While many members of large gene families are incorporated into the chromosome assemblies, the majority of genes excluded from the chromosomes belong to gene families, as these genes are frequently impossible to accurately position.

Conclusion: Now assembled, these chromosomes bring T. cruzi to the same level of organization as its kinetoplastid relatives and have been used as the basis for the T. cruzi genome in TriTrypDB, a trypanosome database of EuPathDB. In addition, they will provide the foundation for analyses such as reverse genetics, where the location of genes and their alleles and/or paralogues is necessary and comparative genome hybridization analyses (CGH), where a chromosome-level view of the genome is ideal.

Show MeSH
Related in: MedlinePlus