rnaQUAST 1.2.0 (reference based) using DE

The DE Quick Start tutorial provides an introduction to basic DE functionality and navigation.

Rationale and background:

rnaQUAST is a tool for evaluating RNA-Seq assemblies using reference genome and gene data database. In addition, rnaQUAST is also capable of estimating gene database coverage by raw reads and de novo quality assessment using third-party software. The following tutorial is reference based assessement of transcripts using rnaQUAST 1.2.0. If you don't have a reference genome then you can use denovo based rnaQUAST 1.2.0 app

Pre-Requisites

1. A CyVerse account. (Register for an CyVerse account here - user.cyverse.org)
2. Input/Outputs 
   1. Transcript file(s) in FASTA format (Mandatory)
   2. Reference genome containing all chromosomes/scaffolds in FASTA format (preferably with *.fasta, *.fa, *.fna, *.ffn or *.frn extension) OR 
      *.txt file containing the one-per-line list of FASTA files with reference sequences (Mandatory)
   3. GTF/GFF gene database file (needs information about parent relations). We recommend to use files downloaded from GENCODE or Ensembl (Optional)
   4. File with forward paired-end reads in FASTQ format.
   5. File with reverse paired-end reads in FASTQ format.
   6. File with single reads in FASTQ format
   7. Output directory to store all results.
3. Options
   
   1.  Run with BLAT alignment tool instead of GMAP.
      
   2.  Run with TopHat tool instead of STAR for analyzing database coverage by reads.
      
   3. Name(s) of assemblies that will be used in the reports separated by space and given in the same order as files with transcripts / alignments. 
   4. Set if transcripts were assembled using strand-specific RNA-Seq data in order to benefit from knowing whether the transcript originated from the + or - strand.
   5.  Do not draw plots (makes rnaQUAST run a bit faster).
   6. Run disable_infer_genes option if your GTF file already contains genes records, otherwise gffutils will fix it. Note that gffutils may work for quite a long time.
   7. Run disable_infer_transcripts if your GTF file already contains transcripts records, otherwise gffutils will fix it. Note that gffutils may work for quite a long time.

Test/sample data:

a. Using rnaQUAST 1.2.0 tool using STAR aligner (default)

Upload the following from /iplant/home/shared/iplantcollaborative/example_data/rnaQUAST.sample.data

1. Input file(s): idba.fasta, spades.311.fasta and Trinity.fasta (transcript files)

2. Input reference genome file: Saccharomyces_cerevisiae.R64-1-1.75.dna.toplevel.fa
3. Input genome annotation file (GFF or GTF): Saccharomyces_cerevisiae.R64-1-1.75.gtf
4. Left Reads: Paired_ends1.fq 
5. Right Reds: Paired_ends2.fq
6. Output folder name - rnaQUAST_output_STAR

and leave the rest of the options as default

b. Using rnaQUAST 1.1.0 tool using TopHat aligner

1. Input file(s): idba.fasta, spades.311.fasta and Trinity.fasta (transcript files)

2. Input reference genome file: Saccharomyces_cerevisiae.R64-1-1.75.dna.toplevel.fa
3. Input genome annotation file (GFF or GTF): Saccharomyces_cerevisiae.R64-1-1.75.gtf
4. Left Reads: Paired_ends1.fq 
5. Right Reds: Paired_ends2.fq
6. Check the "Use TopHat instead of STAR aligner" box
7. Output folder name - rnaQUAST_output_Tophat

and leave the rest of the options as default

Output Reports

The following text files with reports are contained in comparison_output directory and include results for all input assemblies. In addition, these reports are contained in<assembly_label>_output directories for each assembly separately.

database_metrics.txt 
Gene database metrics.

• Genes / Protein coding genes – number of genes / protein coding genes
• Isoforms / Protein coding isoforms – number of isoforms / protein coding isoforms
• Exons / Introns – total number of exons / introns
• Total / Average length of all isoforms, bp
• Average exon length, bp
• Average intron length, bp
• Average / Maximum number of exons per isoform

Coverage by reads. The following metrics are calculated only when --left_reads, --right_reads, --single_reads or --sam options are used (see options for details).

• Database coverage – the total number of bases covered by reads (in all isoforms) divided by the total length of all isoforms.
• x%-covered genes / isoforms / exons – number of genes / isoforms / exons from the database that have at least x% of bases covered by all reads, where x is specified with --lower_threshold / --upper_threshold options (50% / 95% by default).

basic_mertics.txt 
Basic transcripts metrics are calculated without reference genome and gene database.

• Transcripts – total number of assembled transcripts.
• Transcripts > 500 bp
• Transcripts > 1000 bp
• Average length of assembled transcripts
• Longest transcript
• Total length
• Transcript N50 – a maximal number N, such that the total length of all transcripts longer than N bp is at least 50% of the total length of all transcripts.

alignment_metrics.txt 
Alignment metrics are calculated with reference genome but without using gene database. To calculate the following metrics rnaQUAST filters all short partial alignments (see --min_alignmentoption) and attempts to select the best hits for each transcript.

• Transcripts – total number of assembled transcripts.
• Aligned – the number of transcripts having at least 1 significant alignment.
• Uniquely aligned – the number of transcripts having a single significant alignment.
• Multiply aligned – the number of transcripts having 2 or more significant alignments. Multiply aligned transcripts are stored in <assembly_label>.paralogs.fasta file.
• Misassembly candidates reported by GMAP (or BLAT) – transcripts that have discordant best-scored alignment (partial alignments that are either mapped to different strands / different chromosomes / in reverse order / too far away).
• Unaligned – the number of transcripts without any significant alignments. Unaligned transcripts are stored in <assembly_label>.unaligned.fasta file.

Number of assembled transcripts = Unaligned + Aligned = Unaligned + (Uniquely aligned + Multiply aligned + Misassembly candidates reported by GMAP (or BLAT)).

Alignment metrics for non-misassembled transcripts

• Average aligned fraction. Aligned fraction for a single transcript is defined as total number of aligned bases in the transcript divided by the total transcript length.
• Average alignment length. Aligned length for a single transcript is defined as total number of aligned bases in the transcript.
• Average blocks per alignment. A block is defined as a continuous alignment fragment without indels.
• Average block length (see above).
• Average mismatches per transcript – average number of single nucleotide differences with reference genome per transcript.
• NA50 – N50 for alignments.

misassemblies.txt 

• Transcripts – total number of assembled transcripts.
• Misassembly candidates reported by GMAP (or BLAT) – transcripts that have discordant best-scored alignment (partial alignments that are either mapped to different strands / different chromosomes / in reverse order / too far away).
• Misassembly candidates reported by BLASTN – transcripts are aligned to the isoform sequences extracted from the genome using gene database with BLASTN and then transcripts that have partial alignments to multiple isoforms are selected.
• Misassemblies – misassembly candidates confirmed by both methods described above. Using both methods simultaneously allows to avoid considering misalignments that can be caused, for example, by paralogous genes or genomic repeats. Misassembled transcripts are stored in <assembly_label>.misassembled.fasta file.

sensitivity.txt 
Assembly completeness (sensitivity). For the following metrics (calculated with reference genome and gene database) rnaQUAST attempts to select best-matching database isoforms for every transcript. Note that a single transcript can contribute to multiple isoforms in the case of, for example, paralogous genes or genomic repeats. At the same time, an isoform can be covered by multiple transcripts in the case of fragmented assembly or duplicated transcripts in the assembly.

• Database coverage – the total number of bases covered by transcripts (in all isoforms) divided by the total length of all isoforms.
• Duplication ratio – total number of aligned bases in assembled transcripts divided by the total number of isoform covered bases. This metric does not count neither paralogous genes nor shared exons, only real overlaps of the assembled sequences that are mapped to the same isoform.
• Average number of transcripts mapped to one isoform.
• x%-assembled genes / isoforms / exons – number of genes / isoforms / exons from the database that have at least x% captured by a single assembled transcript, where x is specified with --lower_threshold / --upper_threshold options (50% / 95% by default). 95%-assembled isoforms are stored in <assembly_label>.95%assembled.fasta file.
• x%-covered genes / isoforms – number of genes / isoforms from the database that have at least x% of bases covered by all alignments, where x is specified with --lower_threshold / --upper_threshold options (50% / 95% by default).
• Mean isoform assembly – assembled fraction of a single isoform is calculated as the largest number of its bases captured by a single assembled transcript divided by its length; average value is computed for isoforms with > 0 bases covered.
• Mean isoform coverage – coverage of a single isoform is calculated as the number of its bases covered by all assembled transcripts divided by its length; average value is computed for isoforms with > 0 bases covered.
• Mean exon coverage – coverage of a single exon is calculated as the number of its bases covered by all assembled transcripts divided by its length; average value is computed for exons with > 0 bases covered.
• Average percentage of isoform x%-covered exons, where x is specified with --lower_threshold / --upper_threshold options (50% / 95% by default). For each isoform rnaQUAST calculates the number of x%-covered exons divided by the total number of exons. Afterwards it computes average value for all covered isoforms.

specificity.txt 

Assembly specificity. To compute the following metrics we use only transcripts that have at least one significant alignment and are not misassembled.

• Unannotated – total number of transcripts that do not cover any isoform from the database. Unannotated transcripts are stored in <assembly_label>.unannotated.fasta file.
• x%-matched – total number of transcripts that have at least x% covering an isoform from the database, where x is specified with --lower_threshold / --upper_threshold options (50% / 95% by default).
• Mean fraction of transcript matched – matched fraction of a single transcript is calculated as the number of its bases covering an isoform divided by the transcript length; average value is computed for transcripts with > 0 bases matched.
• Mean fraction of block matched – matched fraction of a single block is calculated as the number of its bases covering an isoform divided by the block length; average value is computed for blocks with > 0 bases matched.
• x%-matched blocks – percentage of blocks that have at least x% covering an isoform from the database, where x is specified with --lower_threshold / --upper_threshold options (50% / 95% by default).
• Matched length – total number of transcript bases covering isoforms from the database.
• Unmatched length – total alignment length - Matched length.

relative_database_coverage.txt 
Relative database coverage metrics are calculated only when raw reads (or read alignments) are provided. rnaQUAST uses read alignments to estimate the upper bound of the database coverage and the number of x-covered genes / isoforms / exons (see read coverage) and computes the following metrics:

• Relative database coverage – ratio between transcripts database coverage and reads database coverage.
• Relative x%-assembled genes / isoforms / exons – ratio between transcripts x%-assembled and reads x%-covered genes / isoforms / exons.
• Relative x%-covered genes / isoforms / exons – ratio between transcripts x%-covered and reads x%-covered genes / isoforms / exons.

Detailed output

These files are contained in <assembly_label>_output directories for each assembly separately.

• <assembly_label>.unaligned.fasta – transcripts without any significant alignments.
• <assembly_label>.paralogs.fasta – transcripts having 2 or more significant alignments.

• <assembly_label>.misassembled.fasta – misassembly candidates detected by methods described above. See misassemblies.txt description for details.
• <assembly_label>.correct.fasta – transcripts with exactly 1 significant alignment that do not contain misassemblies.
• <assembly_label>.x%assembled.list – IDs of the isoforms from the database that have at least x% captured by a single assembled transcript, where x is specified by the user with an option --upper_threshold (95% by default).
• <assembly_label>.unannotated.fasta – transcripts that do not cover any isoform from the database.

Plots

The following plots are similarly contained in both comparison_output directory and <assembly_label>_output directories. Please note, that most of the plots represent cumulative distributions and some plots are given in logarithmic scale.

Basic

• transcript_length.png – assembled transcripts length distribution (+ database isoforms length distribution).
• block_length.png – alignment blocks length distribution (+ database exons length distribution).
• x-aligned.png – transcript aligned fraction distribution.
• blocks_per_alignment.png – distribution of number of blocks per alignment (+ distribution of number of database exons per isoform).
• alignment_multiplicity.png – distribution for the number of significant alignment for each multiply-aligned transcript.
• mismatch_rate.png – substitution errors per alignment distribution.
• Nx.png – Nx plot for transcripts. Nx is a maximal number N, such that the total length of all transcripts longer than N bp is at least x% of the total length of all transcripts.
• NAx.png – Nx plot for alignments.

Sensitivity

• x-assembled.png – a histogram in which each bar represents the number of isoforms from the database that have at least x% captured by a single assembled transcript.
• x-covered.png – a histogram in which each bar represents the number of isoforms from the database that have at least x% of bases covered by all alignments.
• x-assembled_exons.png – a histogram in which each bar represents the number of exons from the database that have at least x% captured by a single assembled transcript.
• x-covered_exons.png – a histogram in which each bar represents the number of exons from the database that have at least x% of bases covered by all alignments.
• alignments_per_isoform.png – plot showing number of transcript alignments per isoform

Specificity

• x-matched.png – a histogram in which each bar represents the number of transcripts that have at least x% matched to an isoform from the database.
• x-matched_blocks.png – a histogram in which each bar represents the number of all blocks from all transcript alignments that have at least x% matched to an isoform from the database.