Most of routines specified in this section will output processed clonotype tables and re-normalize individual clonotype frequencies by dividing their read count by the total read count in resulting (filtered/processed) sample. For some of the routines this behavior can be disabled with --save-freqs option. In this case original clonotype frequencies will be carried over from input samples and they will likely not sum to 1.0 in the resulting clonotype table.
Performs frequency-based correction to eliminate erroneous clonotypes. Searches the sample for clonotype pairs that differ by one, two … (up to specified depth) mismatches. In case the ratio of smallest to largest clonotype sizes is lower than the threshold specified as ratio ^ number_of_mismatches correction is performed. Largest clonotype in pair increases its size by the read count of the smaller one and the smaller one is discarded. Note that the original sample is not changed during correction, so all comparisons are performed with original count values and erroneous clonotypes are only removed after search procedure is finished. It is also possible to restrict correction to clonotypes with identical V/J segments using -a option.
Outputs corrected samples to the path specified by output prefix and creates a corresponding metadata file. Will also append corr:[-d option value]:[-r option value]:['vjmatch' or 'all' based on -a option] to ..filter.. metadata column.
Cross-sample contamination can occur at library prep stage, for example sample barcode swithing resulting from PCR chimeras. Those could lead to a high number of artificial shared clonotypes for samples sequenced in the same batch. If no sophisticated library prep method (e.g. paired-end barcoding) is applied, it is highly recommended to filter those before performing any kind of cross-sample analysis.
This routine filters out all clonotypes that have a matching clonotype in a different sample which is -r times more abundant. Clonotype fractions within samples are considered, which is good for dealing with FACS-related contaminations. In case of dealing with cross-sample contaminations in samples coming from the same sequencing lane use --read-based option that tells the routine to compare read counts.
--read-based string If set will compare clonotype read counts. Clonotype fractions in corresponding samples are compared by default.
Outputs filtered samples to the path specified by output prefix and creates a corresponding metadata file. Will also append dec:[-r value] to ..filter.. metadata column.
speeding up computation / decreasing file size and memory footprint.
Outputs sub-samples to the path specified by output prefix and creates a corresponding metadata file. Will also append ds:[-x value] to ..filter.. metadata column.
Filters non-functional (non-coding) clonotypes, i.e. the ones that contain a stop codon or frameshift in their receptor sequence. Those clonotypes do not have any functional role, but they are useful for dissecting and studying the V-(D)-J recombination machinery as they do not pass thymic selection.
Outputs filtered samples to the path specified by output prefix and creates a corresponding metadata file. Will also append ncfilter:[retain or remove based on -e option] to ..filter.. metadata column.
Creates a filter summary file with a ncfilter.summary.txt suffix containing info on the number of unique clonotypes that passed the filtering process, their total frequency and count.
Selects top N clonotypes from the sample. Useful for studying exapanded clonotypes and clonotypes with strong convergent recombination bias, as well as robust computing of unweighted statistics.
Outputs sub-samples to the path specified by output prefix and creates a corresponding metadata file. Will also append top:[-x value] to ..filter.. metadata column.
Selects clonotypes that either have a frequency above the specified threshold and/or constitute more than a specified percent of reads (e.g. quantile threshold of 0.25 will top N clonotypes that in total contain 25% of reads in the sample). Those two filters can be used together or separately by setting either frequency threshold to 0 or quantile threshold to 1.
Outputs filtered samples to the path specified by output prefix and creates a corresponding metadata file. Will also append freqfilter:[-f value]:[-q value] to ..filter.. metadata column.
Retains/filters out all clonotypes found in a given sample S from other samples. Useful when S contains some specific cells of interest e.g. tumor-infiltrating T-cells or sorted tetramer+ T-cells.
Outputs filtered samples to the path specified by output prefix and creates a corresponding metadata file. Will also append asaf:[- if -e, + otherwise]:[-i value] to ..filter.. metadata column.
Filters clonotypes that have V/D/J segments that match a specified segment set.
-n --negative Retain only clonotypes that lack specified V/D/J segments.
-v --v-segments v1,v2,… A comma-separated list of Variable segment names. Non-matching incomplete names will be partially matched.
-d --d-segments d1,d2,… A comma-separated list of Diversity segment names. Non-matching incomplete names will be partially matched.
-j --j-segments j1,j2,… A comma-separated list of Joining segment names. Non-matching incomplete names will be partially matched.
Outputs filtered samples to the path specified by output prefix and creates a corresponding metadata file. Will also append segfilter:[retain or remove based on -e option]:[-v value]:[-d value]:[-j value] to ..filter.. metadata column.
Creates a filter summary file with a segfilter.summary.txt suffix containing info on the number of unique clonotypes that passed the filtering process, their total frequency and count.