FastQC software [49] to check the quality of the reads. Illumina
sequences are aligned to the reference genome using a splice-aware
aligner such as TopHat [50] or HISAT2 [51]. Transcript assembly is
performed using Cufflinks and differentially expressed genes and
transcripts are identified using Cuffdiff [52]. Transcript variants
aligned to the genome are visualized using the Integrative Genomic
Viewer [53]. Open reading frames of lncRNAs are predicted using
the NCBI Open Reading Frame Finder. Coding potential analysis is
performed using two different alignment-free methods: Coding
Potential Calculator 2 [54] and Coding Potential Assessment
Tool [55]. Finally, the annotation for lncRNAs according to the
genomic context is discussed [56].
1. Connect to Galaxy (https://usegalaxy.org/) using your username and password. Enter to the section Get Data on the tools
panel and select Upload File from your computer. Upload the
sequence of your genome of reference in FASTA format and
the file with annotation information in GFF3 format. The files
can be accessed by direct uploading from your computer using
the option Choose File or by downloading from the web, using
the option Paste/Fetch Data. Once appointed the files, click on
the bottom Start. Loaded files appear on the History panel.
2. Upload sequence reads in FASTQ format selecting the tool
Upload file from your computer. Select them and confirm
uploading by clicking the bottom Start.
3. Check the quality of sequence data using the FastQC Read
Quality reports tool located on the tools panel, Genomic File
Manipulation section, folder FASTQ Quality Control. Select
the FASTQ files as input for the analysis. Click the Execute
bottom. The tool produces an output file that contains basic
statistics, per base sequence quality score and per base sequence
content, GC content, N content, sequence length distribution,
sequence duplication levels, and overrepresented sequences.
4. Filter reads based on the quality scores using the Filter by
quality tool located on the tools panel, Genomic File Manipulation section, folder FASTA/FASTQ. Quality score distribution is calculated for each read (cutoff value of quality score of
30 is recommended). The FASTQ Trimmer tool can be used to
trim low-quality ends of reads.
5. Map reads to your reference genome using TopHat or HISAT2
tool located on the tools panel, Genomic Analysis section,
folder RNA-seq. Select the FASTQ file or two FASTQ files
depending on if the library was sequenced by single end or
paired end, and the FASTA file containing the genome
sequence previously uploaded. If your reads are from a
stranded library, choose the appropriate setting under Specify
strand information. A set of parameters can be modified by
464
Soledad Traubenik et al.
sequences are aligned to the reference genome using a splice-aware
aligner such as TopHat [50] or HISAT2 [51]. Transcript assembly is
performed using Cufflinks and differentially expressed genes and
transcripts are identified using Cuffdiff [52]. Transcript variants
aligned to the genome are visualized using the Integrative Genomic
Viewer [53]. Open reading frames of lncRNAs are predicted using
the NCBI Open Reading Frame Finder. Coding potential analysis is
performed using two different alignment-free methods: Coding
Potential Calculator 2 [54] and Coding Potential Assessment
Tool [55]. Finally, the annotation for lncRNAs according to the
genomic context is discussed [56].
1. Connect to Galaxy (https://usegalaxy.org/) using your username and password. Enter to the section Get Data on the tools
panel and select Upload File from your computer. Upload the
sequence of your genome of reference in FASTA format and
the file with annotation information in GFF3 format. The files
can be accessed by direct uploading from your computer using
the option Choose File or by downloading from the web, using
the option Paste/Fetch Data. Once appointed the files, click on
the bottom Start. Loaded files appear on the History panel.
2. Upload sequence reads in FASTQ format selecting the tool
Upload file from your computer. Select them and confirm
uploading by clicking the bottom Start.
3. Check the quality of sequence data using the FastQC Read
Quality reports tool located on the tools panel, Genomic File
Manipulation section, folder FASTQ Quality Control. Select
the FASTQ files as input for the analysis. Click the Execute
bottom. The tool produces an output file that contains basic
statistics, per base sequence quality score and per base sequence
content, GC content, N content, sequence length distribution,
sequence duplication levels, and overrepresented sequences.
4. Filter reads based on the quality scores using the Filter by
quality tool located on the tools panel, Genomic File Manipulation section, folder FASTA/FASTQ. Quality score distribution is calculated for each read (cutoff value of quality score of
30 is recommended). The FASTQ Trimmer tool can be used to
trim low-quality ends of reads.
5. Map reads to your reference genome using TopHat or HISAT2
tool located on the tools panel, Genomic Analysis section,
folder RNA-seq. Select the FASTQ file or two FASTQ files
depending on if the library was sequenced by single end or
paired end, and the FASTA file containing the genome
sequence previously uploaded. If your reads are from a
stranded library, choose the appropriate setting under Specify
strand information. A set of parameters can be modified by
464
Soledad Traubenik et al.