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VTBuilder: a tool for the assembly of multi isoform transcriptomes.

Archer J, Whiteley G, Casewell NR, Harrison RA, Wagstaff SC - BMC Bioinformatics (2014)

Bottom Line: From the simulated reads, VTBuilder constructed 55 transcripts, 50 of which had a greater than 99% sequence similarity to 48 of the SSTs.Unlike other approaches, VTBuilder strives to maintain the relationships between co-evolving sites within the constructed transcripts, and thus increases transcript utility for a wide range of research areas ranging from transcriptomics to phylogenetics and including the monitoring of drug resistant parasite populations.Additionally, improving the quality of transcripts assembled from read data will have an impact on future studies that query these data.

View Article: PubMed Central - PubMed

Affiliation: Department of Parasitology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA2, UK. john.archer.jpa@gmail.com.

ABSTRACT

Background: Within many research areas, such as transcriptomics, the millions of short DNA fragments (reads) produced by current sequencing platforms need to be assembled into transcript sequences before they can be utilized. Despite recent advances in assembly software, creating such transcripts from read data harboring isoform variation remains challenging. This is because current approaches fail to identify all variants present or they create chimeric transcripts within which relationships between co-evolving sites and other evolutionary factors are disrupted. We present VTBuilder, a tool for constructing non-chimeric transcripts from read data that has been sequenced from sources containing isoform complexity.

Results: We validated VTBuilder using reads simulated from 54 Sanger sequenced transcripts (SSTs) expressed in the venom gland of the saw scaled viper, Echis ocellatus. The SSTs were selected to represent genes from major co-expressed toxin groups known to harbor isoform variants. From the simulated reads, VTBuilder constructed 55 transcripts, 50 of which had a greater than 99% sequence similarity to 48 of the SSTs. In contrast, using the popular assembler tool Trinity (r2013-02-25), only 14 transcripts were constructed with a similar level of sequence identity to just 11 SSTs. Furthermore VTBuilder produced transcripts with a similar length distribution to the SSTs while those produced by Trinity were considerably shorter. To demonstrate that our approach can be scaled to real world data we assembled the venom gland transcriptome of the African puff adder Bitis arietans using paired-end reads sequenced on Illumina's MiSeq platform. VTBuilder constructed 1481 transcripts from 5 million reads and, following annotation, all major toxin genes were recovered demonstrating reconstruction of complex underlying sequence and isoform diversity.

Conclusion: Unlike other approaches, VTBuilder strives to maintain the relationships between co-evolving sites within the constructed transcripts, and thus increases transcript utility for a wide range of research areas ranging from transcriptomics to phylogenetics and including the monitoring of drug resistant parasite populations. Additionally, improving the quality of transcripts assembled from read data will have an impact on future studies that query these data. VTBuilder has been implemented in java and is available, under the GPL GPU V0.3 license, from http:// http://www.lstmed.ac.uk/vtbuilder .

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VTBuilders Graphical User Interface (GUI). Green boxes indicate completed steps of the pipeline while grey indicate those yet to be performed. The yellow box shows the step that is currently running while the yellow text provides a brief description of the step currently running. The inset panel displays the setup area that the user is presented with when they initially double click the jar file. The red circle indicates the command that is required if the user wishes to use the software without a GUI via the command line.
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Fig1: VTBuilders Graphical User Interface (GUI). Green boxes indicate completed steps of the pipeline while grey indicate those yet to be performed. The yellow box shows the step that is currently running while the yellow text provides a brief description of the step currently running. The inset panel displays the setup area that the user is presented with when they initially double click the jar file. The red circle indicates the command that is required if the user wishes to use the software without a GUI via the command line.

Mentions: To address the issues associated with current assembly tools we designed VTBuilder (Figure 1), a user-friendly software for the assembly of non-chimeric transcripts. No reference transcriptome is required and the input can be single or paired end read data in FASTQ format. The software can be launched by executing a single jar file at which point the user will be presented with a Graphical User Interface (GUI) (Figure 1: inset) from which the user can interact with the software via the GUI or using the dynamically generated command in a terminal window (Figure 1: inset, red circle). Installing and running VTBuilder is described in a user guide that is available on the project website. VTBuilder implements a six step bioinformatics pipeline that is described in detail within the implementation section. Briefly, (i) Reads are partitioned into broad groups of shared diversity such as protein families. (ii) De novo assembly on each partition is performed to produce a set of guide sequences. (iii) A set of scaffold-like alignments, similar to those used in reference based assembly [45,46], is produced by mapping each read to the guide sequence that it is most similar to; (iv) For each scaffold like alignment a network is created that represents the isoform diversity present; (v) Transcripts are constructed by traversing these networks; and (vi) Transcript expression is calculated by remapping the read data to the constructed transcripts and then counting the reads mapped to each followed by length normalization.Figure 1


VTBuilder: a tool for the assembly of multi isoform transcriptomes.

Archer J, Whiteley G, Casewell NR, Harrison RA, Wagstaff SC - BMC Bioinformatics (2014)

VTBuilders Graphical User Interface (GUI). Green boxes indicate completed steps of the pipeline while grey indicate those yet to be performed. The yellow box shows the step that is currently running while the yellow text provides a brief description of the step currently running. The inset panel displays the setup area that the user is presented with when they initially double click the jar file. The red circle indicates the command that is required if the user wishes to use the software without a GUI via the command line.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4260244&req=5

Fig1: VTBuilders Graphical User Interface (GUI). Green boxes indicate completed steps of the pipeline while grey indicate those yet to be performed. The yellow box shows the step that is currently running while the yellow text provides a brief description of the step currently running. The inset panel displays the setup area that the user is presented with when they initially double click the jar file. The red circle indicates the command that is required if the user wishes to use the software without a GUI via the command line.
Mentions: To address the issues associated with current assembly tools we designed VTBuilder (Figure 1), a user-friendly software for the assembly of non-chimeric transcripts. No reference transcriptome is required and the input can be single or paired end read data in FASTQ format. The software can be launched by executing a single jar file at which point the user will be presented with a Graphical User Interface (GUI) (Figure 1: inset) from which the user can interact with the software via the GUI or using the dynamically generated command in a terminal window (Figure 1: inset, red circle). Installing and running VTBuilder is described in a user guide that is available on the project website. VTBuilder implements a six step bioinformatics pipeline that is described in detail within the implementation section. Briefly, (i) Reads are partitioned into broad groups of shared diversity such as protein families. (ii) De novo assembly on each partition is performed to produce a set of guide sequences. (iii) A set of scaffold-like alignments, similar to those used in reference based assembly [45,46], is produced by mapping each read to the guide sequence that it is most similar to; (iv) For each scaffold like alignment a network is created that represents the isoform diversity present; (v) Transcripts are constructed by traversing these networks; and (vi) Transcript expression is calculated by remapping the read data to the constructed transcripts and then counting the reads mapped to each followed by length normalization.Figure 1

Bottom Line: From the simulated reads, VTBuilder constructed 55 transcripts, 50 of which had a greater than 99% sequence similarity to 48 of the SSTs.Unlike other approaches, VTBuilder strives to maintain the relationships between co-evolving sites within the constructed transcripts, and thus increases transcript utility for a wide range of research areas ranging from transcriptomics to phylogenetics and including the monitoring of drug resistant parasite populations.Additionally, improving the quality of transcripts assembled from read data will have an impact on future studies that query these data.

View Article: PubMed Central - PubMed

Affiliation: Department of Parasitology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA2, UK. john.archer.jpa@gmail.com.

ABSTRACT

Background: Within many research areas, such as transcriptomics, the millions of short DNA fragments (reads) produced by current sequencing platforms need to be assembled into transcript sequences before they can be utilized. Despite recent advances in assembly software, creating such transcripts from read data harboring isoform variation remains challenging. This is because current approaches fail to identify all variants present or they create chimeric transcripts within which relationships between co-evolving sites and other evolutionary factors are disrupted. We present VTBuilder, a tool for constructing non-chimeric transcripts from read data that has been sequenced from sources containing isoform complexity.

Results: We validated VTBuilder using reads simulated from 54 Sanger sequenced transcripts (SSTs) expressed in the venom gland of the saw scaled viper, Echis ocellatus. The SSTs were selected to represent genes from major co-expressed toxin groups known to harbor isoform variants. From the simulated reads, VTBuilder constructed 55 transcripts, 50 of which had a greater than 99% sequence similarity to 48 of the SSTs. In contrast, using the popular assembler tool Trinity (r2013-02-25), only 14 transcripts were constructed with a similar level of sequence identity to just 11 SSTs. Furthermore VTBuilder produced transcripts with a similar length distribution to the SSTs while those produced by Trinity were considerably shorter. To demonstrate that our approach can be scaled to real world data we assembled the venom gland transcriptome of the African puff adder Bitis arietans using paired-end reads sequenced on Illumina's MiSeq platform. VTBuilder constructed 1481 transcripts from 5 million reads and, following annotation, all major toxin genes were recovered demonstrating reconstruction of complex underlying sequence and isoform diversity.

Conclusion: Unlike other approaches, VTBuilder strives to maintain the relationships between co-evolving sites within the constructed transcripts, and thus increases transcript utility for a wide range of research areas ranging from transcriptomics to phylogenetics and including the monitoring of drug resistant parasite populations. Additionally, improving the quality of transcripts assembled from read data will have an impact on future studies that query these data. VTBuilder has been implemented in java and is available, under the GPL GPU V0.3 license, from http:// http://www.lstmed.ac.uk/vtbuilder .

Show MeSH