Genome-annotation: Transcriptome Alignment

Galaxy Australia is capable of performing genome repeat masking and transcriptome alignment with stringtie and transdecoder

This How-to-Guide will describe the steps required to repeat your genome and align transcripts on the Galaxy Australia platform (see Fig 1), developed in consultations between the Bioplatforms Australia Threatened Species Initiative, Galaxy Australia, and the Australian BioCommons.

If you need help, the Galaxy community is both approachable and helpful. Ask them questions!

Quick start guide

1. Login to Galaxy Australia
2. Create a new history
3. Upload your assembled reference genome and raw RNA sequencing reads (can be imported from Bioplatforms Australia Portal)
4. Load and execute workflows, using required options
   • Open Repeat masking workflow
   • Open RNA seq QC and read trimming workflow
   • Open Align reads to find transcripts workflow
   • Open Combine transcripts workflow
   • Open Extract longest transcripts workflow
   • Open Convert outputs workflow
5. Review workflow report and perform additional QC as needed
6. Re-run workflows, or individual tools, as needed

How to cite the workflows

Silver, L., & Syme, A. (2024). Repeat masking - TSI. WorkflowHub. https://doi.org/10.48546/WORKFLOWHUB.WORKFLOW.875.3

Silver, L., & Syme, A. (2024). QC and trimming of RNAseq reads - TSI. WorkflowHub. https://doi.org/10.48546/WORKFLOWHUB.WORKFLOW.876.1

Silver, L., & Syme, A. (2024). Find transcripts - TSI. WorkflowHub. https://doi.org/10.48546/WORKFLOWHUB.WORKFLOW.877.1

Silver, L., & Syme, A. (2024). Combine transcripts - TSI. WorkflowHub. https://doi.org/10.48546/WORKFLOWHUB.WORKFLOW.878.1

Silver, L., & Syme, A. (2024). Extract transcripts - TSI. WorkflowHub. https://doi.org/10.48546/WORKFLOWHUB.WORKFLOW.879.1

Silver, L., & Syme, A. (2024). Convert formats - TSI. WorkflowHub. https://doi.org/10.48546/WORKFLOWHUB.WORKFLOW.880.1

The overall workflow

Further to this, a summary of the different elements of this alignment approach are detailed below:

Process name	Workflow name	Description	Inputs	Outputs
UPLOAD FILES	Not applicable	See the different upload options.	reference genome, Fastq mRNA	Uploaded data!
Repeat Masking	Repeat masking - TSI	Repeat masking of reference genome	Reference genome	FASTA files of hard-masked and soft-masked genomes, Statistic file
RNA seq QC and trimming	QC and trimming of RNAseq reads -TSI	Trimming of fastq files, including a fastqc step	Raw mRNA sequencing files	FASTQC report, Paired read FASTQ file
Align reads to find transcripts	Find transcripts - TSI	Alignment of trimmed FASTQ reads to masked reference genome	(soft) repeat masked reference genome, paired trimmed FASTQ reads	BAM file, GTF file alignment metrics
Combine Transcripts	Combine Transcripts - TSI	Merges individual tissue transcripts to a global transcriptome and predicts coding sequences	GTF file, soft-masked genome, closely related species coding and non-coding sequences	GTF for global transcriptome, FASTA sequences of coding transcripts
Extract Longest Transcripts	Extract Transcripts-TSI	Transdecoder predictions and filtering of transcripts	FASTA sequence of coding transcripts	pep.fasta, cds.fasta and gff3 file of longest isoform transcripts
Convert Outputs	Convert formats - TSI	Converts outputs of transcdecoder to required inputs for FGenesH++ annotation	transdecoder-peptides.fasta, global_nucleotides.fasta	.cdna, .dat and .pro files

In-depth workflow guide

Register and login

1. To register for Galaxy Australia, visit the login page.
2. Click the Register here link, as shown in Fig 2.
3. Complete the registration wizard and click Create.
4. Login to your account!

Upload data file(s)

1. In Galaxy Australia, create a new history and click on Upload Data and choose local files (See Figure 3)
2. Upload your assembled reference genome and raw mRNA transcriptome FASTQ files

Run the Repeat Masking Workflow

1. Make sure you are logged into Galaxy Australia
2. Visit this link to:
   • Retrieve the workflows for Repeat Masking
   • Import into your Galaxy Australia workflows
3. Once you have reached the workflow screen, select the play button for Repeat Masking (See Figure 4)
4. The workflow invoation window will open. Select your reference genome fasta file (Figure 5) and run workflow

Run RNA seq QC and trimming workflow

1. Visit this link to:
   • Retrieve the workflows for QC and trimming of RNAseq reads
   • Import into your Galaxy Australia workflows
2. If each tissue was sequences across multiple lanes, make two datasets, one containing forward reads and one containing reverse reads
3. Once you have reached the workflow screen, select the play button for QC and trimming of RNAseq reads (See Figure 6).
4. Select forward and reverse datasets and run workflow (Figure 7)
5. Check the MultiQC webpage for each trimmed collection and “#table-reads-info-after-trimming” files to quality check trimmed reads

Run align reads to find transcripts workflow

1. Visit this link to:
   • Retrieve the workflows for Align reads to find transcripts
   • Import into your Galaxy Australia workflows
2. Once you have reached the workflow screen, select the play button for Align reads to find transcripts (Fig 8)
3. Select the paired forward and paired reverse trimmed reads and soft-masked reference genome as input (Fig9), ensure you select files tagged with #fastq_out_r1_paired and #fastq_out_r2_paired
4. Check the mapping summary file for each tissue to make sure there are high mapping rates to the genome
5. Make a dataset collection containing gtf files for all tissue transcriptomes

Run Combine Transcripts

1. Visit this link to:
   • Retrieve the workflow for Combine transcripts
   • Import into your Galaxy Australia workflows
2. Once you have reached the workflow screen, select the play button for Combine Transcripts (Fig 10)

3. Search for your species on NCBI Taxonomy to find the most closely related species which has an NCBI RefSeq annotation (Fig 11)

1. Go to the NCBI ftp server and locate the entry for this species (e.g. Corroborree frog RefSeq entry is GCF_028390025.1 and ftp entry is https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/028/390/025/)
2. Download the _cds_from_genomic.fna.gz and pseudo_without_product.fna.gz files to your local computer and upload into Galaxy (Fig 12)

6. Select the gtf collection, soft-masked reference genome, coding sequences and pseudo coding sequences in the combine transcripts workflow
7. In Step 7 of the workflow ensure the masked genome is selected and that in Step 10 of the workflow type “1” in the List of Fields box (Fig 13; Fig 14; Fig 15)

Run Extract Longest Transcripts workflow

1. Visit this link to:
   • Retrieve the workflows for Extract longest transcripts
   • Import into your Galaxy Australia workflows
2. Once you have reached the workflow screen, select the play button for Extract longest transcript (Fig 16)

3. Select the fasta output file from the previous workflow (tagged with #seqs-with-high-coding-prob) as input to the workflow (Fig 17)

4. Also, in workflow step 4, select the most appropriate BUSCO lineage to run on the output file from Transdecoder
5. Check the BUSCO output to ensure a high percentage of complete BUSCO’s are found in the transcriptome

Run Convert Outputs workflow

1. Visit this link to:
   • Retrieve the workflows for Convert outputs
   • Import into your Galaxy Australia workflows
2. Once you have reached the workflow screen, select the play button for Covert outputs (Fig 18)

3. Select the transdecoder peptide fasta file and the text transformed fasta output file from the Combine Transcripts workflow (Fig 19)

4. The output files tagged with #dat, #pro, and #cdna, along with the hard-masked and unmasked reference genome are used as input files for FGenesH++ genome annotation.

Affiliations Contributors