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Reanalyzing the transcriptomic response of A. galli to an anthelmintic drug with Blast2GO

In this analysis, we will reproduce the study that was carried out by Michaela M. Martis et al. in 2017 (doi: http://doi.org/10.1371/journal.pone.0185182) with Blast2GO. 

Introduction

Ascaridia galli is an intestinal parasite which infects a wide range of domestic birds. It is especially important in European farms, where it parasites laying hens and cause some economic problems. The only available treatments are the Benzimidazole derivatives, such as Flubendazole (FLBZ), a group of anthelmintic drugs. However, their overuse could be problematic in the future, as A. galli could develop resistance mechanisms.

The objectives of this transcriptomics study were:

  • To develop a reference transcriptome of A. galli.
  • To understand the response in gene expression before and after the exposure to FLBZ through an RNA-Seq Differential Expression Analysis.
  • To investigate b-tubulin transcripts expression and phylogeny, since several studies have reported the relation between b-tubulin and Benzimidazoles resistance.

 

A. galli

 

 

  1. Data obtention and processing

Firstly, we downloaded raw paired-end sequenced reads from SRA through the fastq-dump tool, from SRA-Toolkit. We obtained six fastq files, one per analyzed sample, and we classified them according to the experimental design, which is described below. Our complete dataset contained about 173 million reads. 

Run
Experimental condition
ERR194831
control
ERR194832
control
ERR194833
control
ERR194834
flubendazole
ERR194835
flubendazole
ERR194836
flubendazole

After downloading, we performed a quality assessment with FastQC, in Blast2GO (in Tools > FastQ Tools > FastQ Quality Check). We observed that the Illumina sequencing adapters had not been removed from the sequences yet.

However, these reads seemed to have good sequence quality, so we did not consider doing quality trimming.

We used Trimmomatic in order to remove the Illumina adapters. Trimmomatic is a command-line software which removes the adapter sequences from the sequenced raw reads. This tool has already been incorporated into Blast2GO (Tools > FastQ Tools > FastQ Preprocessing).

Before Trimmomatic:

Raw reads

After Trimmomatic:

Clean reads

Adapter content (raw reads)  Adapter content (clean reads)

 

 2. De novo transcriptome assembly and completeness assessment

We performed a de novo transcriptome assembly using the cleaned reads obtained from Trimmomatic. We used Trinity, in Blast2GO ("RNA-Seq > RNA-Seq de novo assembly" option) in the non-strand specific mode. After this step, we obtained an assembled transcriptome with 290713 transcripts. 

To assess the completeness of our transcriptome assembly, we searched for nematode orthologs with BUSCO software. We downloaded the orthologs database from its website and we ran the software with our assembly. In the light of the results obtained, we concluded that we had generated a good quality transcriptome: 91.3% of nematode orthologs have, at least, single-copy representation in our assembly, while 5.3% of them were partially found and 3.4% were missing. Furthermore, we achieved similar findings to those resulting from the original study (they found 91.9% complete orthologs, and 3.3% were missing). 

      BUSCO results:

      busco results

 

 3. Open reading frame prediction

TransDecoder results:

 transdecoder results

Once we obtained our transcriptome, we searched for ORFs within the predicted transcripts. We used TransDecoder, a command-line software which extracts and classifies the best ORFs from a transcripts dataset on the basis of Blast and Pfam domain search data. 

121000 ORFs were predicted and classified by TransDecoder according to their completeness: 72.5% of transcripts were complete, 18.8% with a missing start codon, 5.2% with a missing stop codon and 3.5% lacked both start and stop codons. These results were pretty similar to the original ones, where 119000 ORFs were predicted and classified.

 

 4. Functional annotation and InterProScan

We used Blast2GO to perform a functional annotation with default parameters and an InterProScan analysis of the TransDecoder predicted coding transcripts, as they were our new "coding transcriptome". We followed these steps:

  • BlastX against SwissProt database.
  • CloudIPS against HMMPIR, HAMAP and HMMPanther, and IPS against TMHMM. 
  • Mapping and annotation.
  • Merge IPS results to annotation.

Once the annotation step was finished, we obtained a functional annotation for 80,000 transcripts, which means that 67% of the TransDecoder transcripts had a functional annotation. In the original article, only 30,000 transcripts were finally annotated by using the Trinotate suite.

 

 5. Coding transcripts filtering and classification

As TransDecoder predicts ORFs for each isoform, we had to filter this output in order to keep the best ORF per gene. We were working with a simple organism, so we assumed that actually there was only one isoform per gene. We developed a Python script which selects the best isoform ORF according to two criteria: (1) by favouring those with an assigned Pfam domain, and (2) in the absence of predicted domains, or if all the isoform had an assigned Pfam domain, the longest ORF was selected. 

Once we ran the script, we discarded 100,000 sequences and 18,500 isoforms were kept for further analysis.

In addition, our 18,500 transcripts were classified into "high confidence (HC)", if they had Blast results and an ORF predicted; and "low confidence (LC)", if they only met one criterion. 85% of genes were classified as "high confidence", and the remaining ones as "low confidence".

Then, according to the article, we classified transcripts from both datasets into four groups according to their function: "genes with an assigned function", "genes with unknown function", "transposons" or "potential transposons" by examining their description in Blast2GO. Most of the LC genes had an unknown function or they acted as transposable elements, while the HC ones were classified as genes with an assigned function.

LC transcripts:

LC

HC transcripts:

HC

Low confidence transcripts (15%): functional classification. High confidence transcripts (85%): functional classification.

 

 6. Differential Expression Analysis (DEA) 

A DEA was carried out in Blast2GO. First, we created a count table (RNA-Seq > Create Count Table > Transcript-level Quantification) with our filtered coding transcriptome and the clean reads we had generated in step 1. Then, we performed a DEA (RNA-Seq > Pairwise Differential Expression Analysis) with stringent criteria: an adjusted p-value or FDR < 0.005 and a 4-fold change, and we used the experimental design described above to visualize the differentially expressed genes. We detected 79 up- and 112 down-regulated genes, while the authors had found 110 up- and 153 down-regulated ones. 

Heat map:

dea heatmap2

Volcano plot:

volcano plot

Diff. Expression Analysis: heat map. Diff. Expression Analysis: volcano plot.

Then, we searched for up-regulated genes related to stress or drug resistance by using the Blast2GO description filter, and we obtained similar results to those reported by the authors. We found two candidates:

  • Multidrug resistance protein (PGP). A member of the ATP-binding cassette superfamily. The resistance is associated with a decrease in drug accumulation and an increase in drug efflux.
  • Heat shock protein (HSP). A subset of a group of proteins produced in response to exposure to a range of stressful conditions.

Another gene we found was phosphofructokinase (PFK), an enzyme that serves as a key regulatory step in the glycolysis pathway. This finding suggests that glycolysis could be affected by the exposure to FLBZ.

Within down-regulated genes, we found:

  • Glutamate dehydrogenase (GDH). It is an enzyme with a key role in energy homeostasis in nematodes, with reactions involved in ATP production.
  • Cytochrome P450. It is involved in ATP production via oxidative phosphorylation.

Both findings suggest that ATP production is affected by FLBZ. 

 

 7. Enrichment analysis. Fisher's Exact Test

An enrichment analysis was performed with Blast2GO (Analysis > Enrichment analysis (Fisher's Exact Test)). The up-regulated genes analysis showed no results after the enrichment, but we found an interesting overrepresented function within down-regulated genes. The glucuronate metabolism was overrepresented because of the importance of the UDP-glucuronosyltransferase (UGT) enzyme. This enzyme catalyzes the glucuronidation reaction, involving the addition of a glucuronic acid to some xenobiotics. It is an important pathway for the elimination of the most frequent drugs.

This excretion pathway seems to be down-regulated in flubendazole-treated organisms.

      Enrichment results:

      glucuronic

 

 8. Candidate transcripts responsible for anthelmintic resistance: b-tubulin phylogenetic analysis

B-tubulins are cytoskeletal proteins which are suspected to be related to benzimidazoles resistance, as these drugs seem to exert selection pressure in some b-tubulin isoforms. We performed a phylogeny in order to know how identified b-tubulin transcripts from A. galli were related to other tubulins from closely related species.

We downloaded some b-tubulin sequences from C. elegans, A. suum, A. galli, P. equorum and H. contortus as related species. In addition, we obtained a b-tubulin sequence from a remote species, T. spiralis, which was used as an outgroup sequence, to root the tree. Then, we identified four b-tubulin candidates in our transcriptome by using the Blast2GO filters (but none of them were up or down-regulated).

After that, we aligned both the candidates and the downloaded sequences using MAFFT in its "L-INS-I" parameter to perform an accurate local alignment. The output was used to calculate a b-tubulins phylogeny with the PhyML web server. We selected a Bayesian inference and we set the Bootstrap parameter to 50. 

The results we obtained suggest that A. galli b-tubulin candidates are different from the C. elegans and H. contortus b-tubulins, so we concluded that the C. elegans resistance mechanisms may have more in common with strongylid worms than with ascarids. According to the article, these findings suggest that further investigation is required to confirm the exact mechanism involved in the drug binding and selection in ascarids, including A. galli.

Phylogeny results:

phylog


 Conclusions

  • Our de novo transcriptome assembly and the differential expression analysis provided some information about the A. galli response to flubendazole.
  • We used BUSCO, a software which offers information about the assembly quality and completeness. It is an easy-to-use tool and provides a good method to validate an assembly.
  • Blast2GO allowed us to assembly the A. galli transcriptome, to annotate it, to identify the description of up and down regulated genes and to perform an enrichment analysis.
  • We identified up-regulated genes associated with stress conditions, with drug excretion and with the glycolysis pathway.
  • Down-regulated genes were associated with ATP and energy production. 
  • Contrary to what was expected, our enrichment analysis showed that the glucuronate metabolism, a drug elimination pathway, is overrepresented among down-regulated genes.
  • Our phylogenetic analysis of b-tubulin sequences, which was carried out with MAFFT and PhyML, suggested that these proteins might not be involved in the development of drug resistance.

 

 References

  • Martis MM, Tarbiat B, Tydén E, Jansson DS, Höglund J (2017) RNA-Seq de novo assembly and differential transcriptome analysis of the nematode Ascaridia galli in relation to in vivo exposure to flubendazole. PLoS ONE 12(11): e0185182.
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