Output files
Quality check will be performed on the raw fastq file using NanoPlot which it a tool that can be used to produce general quality metrics e.g. quality score distribution, read lengths and other general stats. A NanoPlot-report.html file will be saved under the SampleName/qc/nanoplot folder with the prefix raw. This report displays 6 plots as well as a table of summary statistics.
Example of output plots:
A preprocessed fastq file will be saved in the SampleName/preprocessing output directory which will minimally have its read names trimmed after the first whitespace, for compatiblity purposes with all downstream tools. This fastq file might be additionally trimmed of adapters and/or filtered based on quality and length (if PoreChopABI and/or Chopper were run).
After quality/length trimming, a PoreChopABI log will be saved under the SampleName/preprocessing/porechop folder.
After adapter trimming, a Chopper log file will be saved under the SampleName/preprocessing/chopper folder.
If adapter trimming and/or quality/length trimming is performed, a second quality check will be performed on the processsed fastq file and a NanoPlot-report.html file will be saved under the SampleName/qc/nanoplot folder with the prefix filtered.
If host filtering of reads is performed, an unaligned.fastq.gz file and an unaligned_reads_count.txt file listing the remainder read count will be saved under the SampleName/preprocessing/host_filtering folder.
If the adapter trimming, the quality filtering and/or the host filtering options have been run, a QC report called run_qc_report_YYYYMMDD-HHMMSS.txt will be saved under the qc_report folder
Example of report:
| Sample | raw_reads | quality_filtered_reads | host_filtered_reads | percent_quality_filtered | percent_host_filtered |
|---|---|---|---|---|---|
| MT010 | 315868 | 315081 | 200632 | 99.75 | 63.52 |
After running Kraken2, six files will be saved under the SampleName/read_classification/kraken folder. The kraken2 standard output (ie SampleName.kraken2) taxonomically classifies each sequence. Check the manual for details about each table field.
The kraken_report is tab-delimited, with one line per taxon. The fields of the output, from left-to-right, are as follows:
- Percentage of reads covered by the clade rooted at this taxon
- Number of reads covered by the clade rooted at this taxon
- Number of reads assigned directly to this taxon
- Number of minimizers in read data associated with this taxon
- An estimate of the number of distinct minimizers in read data associated with this taxon
- A rank code, indicating (U)nclassified, (D)omain, (K)ingdom, (P)hylum, (C)lass, (O)rder, (F)amily, (G)enus, or (S)pecies. Taxa that are not at any of these 10 ranks have a rank code that is formed by using the rank code of the closest ancestor rank with a number indicating the distance from that rank. E.g., "G2" is a rank code indicating a taxon is between genus and species and the grandparent taxon is at the genus rank. All other ranks are simply '-'.
- NCBI taxonomy ID number
- Indented scientific name
The scientific names are indented using space, according to the tree structure specified by the taxonomy.
Unclassified reads are saved in a SampleName_unclassified.fastq file.
The Bracken output file (SampleName_bracken_report.txt) is tab delimited and the fields of the output, from left-to-right, are as follows:
- Species name
- Taxonomy ID
- Taxonomy level (S=Species, G=Genus, O=Order, F=Family, P=Phylum, K=Kingdom)
- Number of Kraken Assigned Reads
- Number of Added Reads with Abundance Reestimation
- Total number of Reads after Abundance Reestimation
- Fraction of Total Reads
The SampleName_bracken_report_viral.txt will only retain the virus and viroid species from the Bracken output.
Finally, a filtered output SampleName_bracken_report_viral_filtered.txt in which the viral reads for a given detection represent a fraction of at least 0.0001 of the total reads will be retained.
After running kaiju, six files will be saved under the SampleName/read_classification/kaiju folder.
The kaiju standard output (ie SampleName_kaiju_name.tsv) taxonomically classifies each sequence. The output is tab delimited and the fields of the output, from left-to-right, are as follows:
- either C or U, indicating whether the read is classified or unclassified.
- name of the read
- NCBI taxon identifier of the assigned taxon
- the length or score of the best match used for classification
- the taxon identifiers of all database sequences with the best match
- the accession numbers of all database sequences with the best match
- matching fragment sequence(s)
- Taxon id name
NB: Since the nr database aggregates multiple genes of identical sequences, only the first accession number for each sequence in the nr source file is kept in Kaiju's database and therefore also in the output file.
The number of taxon identifiers (column 5) and accession numbers (column 5) is limited to 20 entries each in order to reduce large outputs produced by highly abundant protein sequences in nr, e.g. from HIV.
A krona output (SampleName_kaiju.krona) is also generated.
The program kaiju2table converts Kaiju's output file(s) into a summary table at the species level. It consists of 4 columns:
- Sample file name
- percent reads matching this taxon id
- taxon_id
- taxon_name
The SampleName_kaiju_summary_viral.tsv only retains viral detections (which are retrieved using a search for the following terms: virus, viroid, viricota, viridae, viriform, virales, virinae, viricetes, virae and viral) Finally in the filtered output SampleName_kaiju_summary_viral.tsv, only detections whose predicted abundance represent a fraction of at least 0.05 of the total reads will be retained.
If a direct blast homology search of the reads was performed against a database, the results will be saved under the SampleName/read_classification/homology_search folder. All the top hits derived for each contig are listed under the file SampleName_read_classification_blastn_top_hits.txt. This file contains the following 26 columns:
- qseqid
- sgi
- sacc
- length
- pident
- mismatch
- gapopen
- qstart
- qend
- qlen
- sstart
- send
- slen
- sstrand
- evalue
- bitscore
- qcovhsp
- stitle
- staxids
- qseq
- sseq
- sseqid
- qcovs
- qframe
- sframe
- species
For a blast search against NCBI NT, if a read matches at least 90% of its length to a virus or viroid as the top blast hit, they will be listed under the SampleName_read_classification_blastn_top_viral_hits.txt file. If the search is against a local viral database, the match has to cover 95% of its length. Coverage is derived using qcovs).
If multiple reads are recovered for the same viral species, only the best hit will be listed under SampleName_assembly_blastn_top_viral_spp_hits.txt. Selection of the best hit is based on e-value followed by length.
The SampleName_assembly_viral_spp_abundance.txt here will list the number of reads recovered for each viral species.
In the example below, 200 reads were recovered matching to the Tomato spotted wilt orthotospovirus:
species Count
Tomato spotted wilt orthotospovirus 200
Finally the SampleName_assembly_queryid_list_with_viral_match.txt will list each unique accession IDs detected in the sample, the viral species they correspond to, and the number of reads matching to it, and their IDs.
We can see from the example above, that the reads matching to tomato spotted wilt orthotospovirus correspond to 2 different accession numbers matching to 2 separate segments (L and E) of the virus. there are 2 reads matching to OM112200 and 3 reads matching to OM112202.
species sacc count qseqid
Tomato spotted wilt orthotospovirus OM112200 2 ['415df728-dbe8-47a8-afba-e15870adfa5e', 'c443fc2e-6bbe-433d-bfdf-6fa7411ab14f']
Tomato spotted wilt orthotospovirus OM112202 3 ['ee4ccc30-b9fd-4c9b-8b0c-5a37059b539b', '20f389f9-a547-4c43-b0b7-5b0f902b408d', 'ead56ab2-328b-4785-a29a-9ae386ad418b']
In this mode, the assembly created by Canu will be saved under the SampleName/assembly/canu folder. In the contig headers, the length of each contig and the number of reads that contributed to it are specified.
The canu log is also saved under the same folder.
A log example of a successful Canu assembly is included below:
-- Finished on Wed Oct 25 22:37:27 2023 (furiously fast) with 50236.869 GB free disk space
----------------------------------------
-- Finished stage 'generateOutputs', reset canuIteration.
--
-- Assembly 'MT483' finished in 'MT483'.
--
-- Summary saved in 'MT483.report'.
--
-- Sequences saved:
-- Contigs -> 'MT483.contigs.fasta'
-- Unassembled -> 'MT483.unassembled.fasta'
--
-- Read layouts saved:
-- Contigs -> 'MT483.contigs.layout'.
--
-- Bye.
Similarly, a fasta file containing the flye assembly and a log of the assembly process will be saved under SampleName/assembly/flye If the Flye assembly ran succesfully it will output some assembly statistics at the end of its log, as per the example below:
[2023-10-30 11:56:06] root: INFO: Assembly statistics:
Total length: 105982
Fragments: 38
Fragments N50: 2768
Largest frg: 4361
Scaffolds: 3
Mean coverage: 89
If a final primer check was performed, then a SampleName_assembly_filtered.fa will be saved under the SampleName/assembly folder along with the log of the cutadapt filtering step.
If a blast homology search of the contigs was performed against a database, the results will be saved under the SampleName/assembly/blastn folder. All the top hits derived for each contig are listed under the file SampleName_assembly_blastn_top_hits.txt. This file contains the following 26 columns:
- qseqid
- sgi
- sacc
- length
- pident
- mismatch
- gapopen
- qstart
- qend
- qlen
- sstart
- send
- slen
- sstrand
- evalue
- bitscore
- qcovhsp
- stitle
- staxids
- qseq
- sseq
- sseqid
- qcovs
- qframe
- sframe
- species
For a blast search against NCBI NT, if a contig sequence matches at least 90% of its length to a virus or viroid as the top blast hit, they will be listed under the SampleName_assembly_blastn_top_viral_hits.txt file. If the search is against a local viral database, the match has to cover 95% of its length. Coverage is derived using qcovs).
If multiple contigs are recovered for the same viral species, only the best hit will be listed under SampleName_assembly_blastn_top_viral_spp_hits.txt. Selection of the best hit is based on e-value followed by length.
The SampleName_assembly_viral_spp_abundance.txt here will list the number of contigs recovered for each viral species.
In the example below, 2 contigs were recovered matching to the Tomato spotted wilt orthotospovirus:
species Count
Tomato spotted wilt orthotospovirus 2
Finally the SampleName_assembly_queryid_list_with_viral_match.txt will list each unique accession IDs detected in the sample, the viral species they correspond to, and the number of contigs matching to it, and their IDs.
We can see from the example above, that the 2 contigs matching to tomato spotted wilt orthotospovirus correspond to 2 different accession numbers matching to 2 separate segments (L and E) of the virus.
species sacc count qseqid
Tomato spotted wilt orthotospovirus OM112200 1 contig_3
Tomato spotted wilt orthotospovirus OM112202 1 contig_4
In this mode, the output from Rattle will be saved under clustering/rattle/SampleName_transcriptome.fq. The number of reads contributing to each clusters is listed in the header. The amplicon of interest is usually amongst the most abundant clusters (i.e. the ones represented by the most reads). A fasta file of the clusters created by Rattle will be saved under the SampleName/clustering/rattle/SampleName_rattle.fasta file. This will only retain the names of the clusters. The final cap3-collapsed clusters can be found in the SampleName/clustering/cap3/SampleName_clustering.fasta file. If cap3 collapsed clusters, they will be referred as Contig_number in the fasta file. Uncollapsed clusters names will remain unaltered (i.e. cluster_number).
In this mode, the reads are alighned directly to a reference file to generate an indexed bam file which will be saved under SampleName/mapping/SampleName_aln.sorted.bam. A SampleName/coverage.txt file is also included which provides a table of coverage as a tabulated text file. Please refer to Samtools coverage for detailed description.
The tabulated form uses the following headings:
| headings | description |
|---|---|
| rname | Reference name / chromosome matching the headers provided in the fasta file |
| startpos | Start position |
| endpos | End position (or sequence length) |
| numreads | Number reads aligned to the region (after filtering) |
| covbases | Number of covered bases with depth >= 1 |
| coverage | Percentage of covered bases [0..100] |
| meandepth | Mean depth of coverage |
| meanbaseq | Mean baseQ in covered region |
| meanmapq | Mean mapQ of selected reads |
An example of table is included below:
| #rname | startpos | endpos | numreads | covbases | coverage | meandepth | meanbaseq | meanmapq |
|---|---|---|---|---|---|---|---|---|
| AobVX | 1 | 6803 | 18893 | 6803 | 100 | 4232.75 | 22.3 | 59.1 |
An ASCII-art histogram called SampleName_histogram is also available to visualise coverage along the genome. The same statistics reported in the coverage.txt file are found on the right hand side of the histogram.
An example is provided below:
AobVX (6.8Kbp)
> 90.00% |::::::::::::::::::::::::::::::::::::::::::::::::::| Number of reads: 18893
> 80.00% |::::::::::::::::::::::::::::::::::::::::::::::::::| (7 filtered)
> 70.00% |::::::::::::::::::::::::::::::::::::::::::::::::::| Covered bases: 6.8Kbp
> 60.00% |::::::::::::::::::::::::::::::::::::::::::::::::::| Percent covered: 100%
> 50.00% |::::::::::::::::::::::::::::::::::::::::::::::::::| Mean coverage: 4.23e+03x
> 40.00% |::::::::::::::::::::::::::::::::::::::::::::::::::| Mean baseQ: 22.3
> 30.00% |::::::::::::::::::::::::::::::::::::::::::::::::::| Mean mapQ: 59.1
> 20.00% |::::::::::::::::::::::::::::::::::::::::::::::::::|
> 10.00% |::::::::::::::::::::::::::::::::::::::::::::::::::| Histo bin width: 136bp
> 0.00% |::::::::::::::::::::::::::::::::::::::::::::::::::| Histo max bin: 100%
1 1.4K 2.7K 4.1K 5.4K 6.8K
results
├── MT001
│ ├── host_filtering
│ │ ├── MT001_unaligned.fastq.gz
│ │ └── MT001_unaligned_reads_count.txt
│ ├── preprocessing
│ │ ├── MT001_preprocessed.fastq.gz
│ │ └── porechop
│ │ └── MT001_porechop.log
│ ├── qc
│ │ └── nanoplot
│ │ ├── MT001_filtered_NanoPlot-report.html
│ │ └── MT001_raw_NanoPlot-report.html
│ ├── assembly
│ │ ├── blastn
│ │ │ ├── MT001_assembly_blastn_top_hits.txt
│ │ │ ├── MT001_assembly_blastn_top_viral_hits.txt
│ │ │ ├── MT001_assembly_blastn_top_viral_spp_hits.txt
│ │ │ ├── MT001_assembly_queryid_list_with_viral_match.txt
│ │ │ └── MT001_assembly_viral_spp_abundance.txt
│ │ ├── canu
│ │ │ ├── MT001_canu_assembly.fasta
│ │ │ └── MT001.canu.log
│ │ ├── MT001_canu_assembly_filtered.fa
│ │ └── MT001_cutadapt.log
│ └── read_classification
│ ├── bracken
│ │ ├── MT001_bracken_report.txt
│ │ ├── MT001_bracken_report_viral_filtered.txt
│ │ ├── MT001_bracken_report_viral.txt
│ │ ├── MT001.kraken2
│ │ ├── MT001_kraken_report.txt
│ │ └── MT001_unclassified.fastq
│ ├── homology_search
│ │ ├── MT001_read_classification_blastn_top_hits.txt
│ │ ├── MT001_read_classification_blastn_top_viral_hits.txt
│ │ ├── MT001_read_classification_blastn_top_viral_spp_hits.txt
│ │ ├── MT001_read_classification_queryid_list_with_viral_match.txt
│ │ └── MT001_read_classification_viral_spp_abundance.txt
│ └── kaiju
│ ├── MT001_kaiju.krona
│ ├── MT001_kaiju_name.tsv
│ ├── MT001_kaiju_summary.tsv
│ ├── MT001_kaiju_summary_viral_filtered.tsv
│ ├── MT001_kaiju_summary_viral.tsv
│ └── MT001_krona.html
└── qc_report
└── run_qc_report_20231009-114823.txt