Output Files

For this example we will use the directory 03_extractions previously created with the extract module. We run the following Captus command to collect markers across samples and align them:

captus_assembly align align -e 03_extractions_CAP/ -o 04_alignments_CAP -m ALL -f ALL

After the run is finished we should see a new directory called 04_alignments with the following structure and files:

Alignments Alignments

Alignment directory Alignment directory

1. 01_unaligned

This directory contains the unaligned FASTA files corresponding to each marker that were gathered from the extractions directory. The files are organized in subdirectories, first by marker type and then by format.

2. 02_untrimmed

This directory contains the aligned FASTA files corresponding to each file in the 01_unaligned directory. The files are organized in subdirectories, first by filtering strategy, then by marker type, and finally by format. The subdirectory structure is identical to the one inside the 03_trimmed directory (see 4 to 15 below). Untrimmed alignments Untrimmed alignments

3. 03_trimmed

All the files present in the 02_untrimmed directory are trimmed using ClipKIT which removes columns that are mostly empty (see options --clipkit_algorithm, --clipkit_gaps), then Captus removes sequences that are too short after trimming (--min_coverage). The files are organized in subdirectories, first by filtering strategy, then by marker type, and finally by format. The subdirectory structure is identical to the one inside the 02_untrimmed directory (see 4 to 15 below). Trimmed alignments Trimmed alignments

4. 01_unfiltered_w_refs

This directory contains the alignments before performing any filtering. All the reference sequences selected by at least a sample will be present as well as all the paralogs per sample. The files are organized in subdirectories, first by marker type and then by format.

5. 02_naive_w_refs

This directory contains the alignments where paralogs have been filtered by the naive method, which consists in simply keeping the best hit per sample (hit ranked as 00). All the reference sequences selected by at least a sample will still be present. The files are organized in subdirectories, first by marker type and then by format. Naive paralog filter Naive paralog filter

6. 03_informed_w_refs

This directory contains the alignments where paralogs have been filtered by the informed method. Under this strategy, Captus compares every copy to the most commonly used reference sequence (sequence ABCD-3400 in the figure) and retains the copy with the highest similarity to that reference, regardless of its paralog ranking (in the figure, Sample1 and Sample4 whose selected copies had paralog rankings of 01 and 02 respectively). All the reference sequences selected by at least a sample will still be present. The files are organized in subdirectories, first by marker type and then by format. Informed paralog filter Informed paralog filter

7. 04_unfiltered, 05_naive, 06_informed

These contain equivalent alignments to directories 01_unfiltered_w_refs, 02_naive_w_refs, and 03_informed_w_refs respectively, but excluding the reference sequences. In most cases you will estimate phylogenies from the trimmed versions of these alignments.

8. 01_coding_NUC, 02_coding_PTD, 03_coding_MIT

These directories contain the aligned coding markers from the NUClear, PlasTiDial, and MITochondrial genomes respectively.
The alignments are presented in four formats: protein sequence (coding_AA), coding sequence in nucleotide (coding_NT), exons and introns concatenated (genes), and the concatenation of exons and introns flanked by a fixed length of sequence (genes_flanked):

Protein extraction formats Protein extraction formats

9. 01_AA

This directory contains the protein alignments (AA in the figure above) of the extracted markers gathered across samples. One FASTA file per marker, with extension .faa.

10. 02_NT

This directory contains the alignments of coding sequence in nucleotides (NT in the figure above) of the extracted markers gathered across samples. One FASTA file per marker, with extension .fna.

11. 03_genes

This directory contains the alignments of gene sequence (exons + introns) in nucleotides (GE in the figure above) of the extracted markers gathered across samples. One FASTA file per marker, with extension .fna.

12. 04_genes_flanked

This directory contains the alignments of flanked gene sequence in nucleotides (GF in the figure above) of the extracted markers gathered across samples. One FASTA file per marker, with extension .fna.

13. 04_misc_DNA, 05_clusters

These directories contain the aligned miscellaneous DNA markers, either from a DNA custom set of references or from the CLusteRing resulting from using the option --cluster_leftovers during the extraction step.
The alignments are presented in two formats: matching DNA segments (matches), and the matched segments including flanks and other intervening segments not present in the reference (matches_flanked).

Miscellaneous DNA extraction formats Miscellaneous DNA extraction formats

14. 01_matches

This directory contains the alignments of DNA sequence matches (MA in the figure above) for the extracted markers gathered across samples. One FASTA file per marker, with extension .fna.

15. 02_matches_flanked

This directory contains the alignments of DNA sequence matches (MF in the figure above) including flanks and intervening segments not present in the references for the extracted markers gathered across samples. One FASTA file per marker, with extension .fna.

16. captus-assembly_align.paralogs.tsv

A tab-separated-values table recording which copy was selected during the informed filtering of paralogs.

Column Description
marker_type Type of marker. Possible values are NUC, PTD, MIT, DNA, or CLR.
format_filtered Alignment format used for identity comparison and filtering. For protein references, if the reference is provided both in aminoacids and nucleotides, the nucleotide format is used.
locus Name of the locus.
ref Name of most common the reference observed in the alignment.
sample Name of the sample.
hit Paralog ranking.
sequence Name of the sequence as it appears in the alignments.
length Lenght of the sequence in aminoacids if format_filtered is AA, or in nucleotides if format_filtered is NT
identity Identity of the sequence to the ref sequence, over the overlapping segment, ignoring internal gaps.
paralog_score (length / length of ref) * (identity / 100), the highest score decides the selected copy.
accepted Whether the copy is accepted (TRUE) or not (FALSE).

17. captus-assembly_align.alignments.tsv

A tab-separated-values table recording alignment statistics for each of the alignments produced.

Column Description
path Absolute path to the alignment file.
trimmed Whether the alignment has been trimmed (TRUE) or not (FALSE).
paralog_filter Filtering strategy applied to the file. Possible values are unfiltered, naive, or informed.
with_refs Whether the file still contains the reference sequences (TRUE) or they have been removed (FALSE).
marker_type Type of marker. Possible values are NUC, PTD, MIT, DNA, or CLR.
format Alignment format. Possible values are AA,NT,GE,GF,MA,MF.
locus Name of the locus.
seqs Number of sequences in the alignment.
samples Number of samples represented in the alignment. The number can be smaller than seqs if the alignment has paralogs.
avg_copies seqs / samples
sites Alignment length.
informative Number of parsimony-informative-sites in the alignment.
informativeness (informative / sites) * 100
uninformative constant + singleton
constant Number of invariant sites in the alignment.
singleton Number of sites with variaton in a single sequence.
paterns Number of unique columns, for a detailed explanation see IQ-TREE’s F.A.Q.: What are the differences between alignment columns/sites and patterns?.
avg_pid Average pairwise identity between sequences in the alignment.
missingness Proportion of missing data (-, N, X, ?, ., ~) in the alignment.
gc
gc_codon_p1
gc_codon_p2
gc_codon_p3

18. captus-assembly_align.samples.tsv

A tab-separated-values table recording sample statistics across the different filtering and trimming stages, as well as marker types and formats.

Column Description
sample Sample name.
stage_marker_format Trimming stage / Filtering strategy / Marker type / Format.
locus Name of the locus.
len_total
len_gapped
len_ungapped
cov_gapped Coverage of the sequence relative to alignment length, including internal gaps.
cov_ungapped Coverage of the sequence relative to alignment length, excluding internal gaps.
ambigs
pct_ambig Percentage of ambiguities in the sequence, excluding gaps.
gc
gc_codon_p1
gc_codon_p2
gc_codon_p3
num_copies Number of copies in the alignment.

19. captus-assembly_align.astral-pro.tsv

ASTRAL-Pro requires a tab-separated-values file for mapping the names of the paralog sequence names (first column) to the name of the sample (second column). Captus produces this file automatically.

GenusA_speciesA_CAP	GenusA_speciesA_CAP
GenusA_speciesA_CAP__00	GenusA_speciesA_CAP
GenusA_speciesA_CAP__01	GenusA_speciesA_CAP
GenusA_speciesA_CAP__02	GenusA_speciesA_CAP
GenusA_speciesA_CAP__03	GenusA_speciesA_CAP
GenusA_speciesA_CAP__04	GenusA_speciesA_CAP
GenusA_speciesA_CAP__05	GenusA_speciesA_CAP
GenusB_speciesB_CAP	GenusB_speciesB_CAP
GenusB_speciesB_CAP__00	GenusB_speciesB_CAP
GenusB_speciesB_CAP__01	GenusB_speciesB_CAP
GenusB_speciesB_CAP__02	GenusB_speciesB_CAP
GenusC_speciesC_CAP	GenusC_speciesC_CAP
GenusC_speciesC_CAP__00	GenusC_speciesC_CAP
GenusC_speciesC_CAP__01	GenusC_speciesC_CAP
GenusC_speciesC_CAP__02	GenusC_speciesC_CAP
GenusC_speciesC_CAP__03	GenusC_speciesC_CAP
GenusC_speciesC_CAP__04	GenusC_speciesC_CAP
GenusC_speciesC_CAP__05	GenusC_speciesC_CAP
GenusD_speciesD_CAP	GenusD_speciesD_CAP
GenusD_speciesD_CAP__00	GenusD_speciesD_CAP
GenusD_speciesD_CAP__01	GenusD_speciesD_CAP
GenusD_speciesD_CAP__02	GenusD_speciesD_CAP
GenusD_speciesD_CAP__03	GenusD_speciesD_CAP
GenusD_speciesD_CAP__04	GenusD_speciesD_CAP
GenusD_speciesD_CAP__05	GenusD_speciesD_CAP

20. captus-assembly_align.report.html

This is the final Aligment report, summarizing alignment statistics across all processing stages, marker types, and formats.

21. captus-assembly_align.log

This is the log from Captus, it contains the command used and all the information shown during the run. Even if the option --show_more was disabled, the log will contain all the extra detailed information that was hidden during the run.

Created by Edgardo M. Ortiz (06.08.2021)
Last modified by Edgardo M. Ortiz (31.05.2022)