| samtools-ampliconstats(1) | Bioinformatics tools | samtools-ampliconstats(1) |
samtools-ampliconstats - produces statistics from amplicon sequencing alignment file
samtools ampliconstats [options] primers.bed in.sam|in.bam|in.cram...
samtools ampliconstats collects statistics from one or more input alignment files and produces tables in text format. The output can be visualized graphically using plot-ampliconstats.
The alignment files should have previously been clipped of primer sequence, for example by "samtools ampliconclip" and the sites of these primers should be specified as a bed file in the arguments. Each amplicon must be present in the bed file with one or more LEFT primers (direction "+") followed by one or more RIGHT primers. For example:
MN908947.3 1875 1897 nCoV-2019_7_LEFT 60 + MN908947.3 1868 1890 nCoV-2019_7_LEFT_alt0 60 + MN908947.3 2247 2269 nCoV-2019_7_RIGHT 60 - MN908947.3 2242 2264 nCoV-2019_7_RIGHT_alt5 60 - MN908947.3 2181 2205 nCoV-2019_8_LEFT 60 + MN908947.3 2568 2592 nCoV-2019_8_RIGHT 60 -
Ampliconstats will identify which read belongs to which amplicon. For purposes of computing coverage statistics for amplicons with multiple primer choices, only the innermost primer locations are used.
A summary of output sections is listed below, followed by more detailed descriptions.
File specific sections start with both the section key and the filename basename (minus directory and .sam, .bam or .cram suffix).
Note that the file specific sections are interleaved, ordered first by file and secondly by the file specific stats. To collate them together, use "grep" to pull out all data of a specific type.
The combined sections (C*) follow the same format as the file specific sections, with a different key. For simplicity of parsing they also have a filename column which is filled out with "COMBINED". These rows contain stats aggregated across all input files.
This section is once per file and includes summary information to be utilised for scaling of plots, for example the total number of amplicons and files present, tool version number, and command line arguments. The second column is the filename or "COMBINED". This is followed by the reference name (unless single-ref mode is enabled), and the summary statistic name and value.
The AMPLICON section is a reformatting of the input BED file. Each line consists of the reference name (unless single-ref mode is enable), the amplicon number and the start-end coordinates of the left and right primers. Where multiple primers are available these are comma separated, for example 10-30,15-40 in the left primer column indicates two primers have been multiplex together covering genome coordinates 10-30 inclusive and 14-40 inclusively.
This section consists of summary counts for the entire set of input files. These may be useful for automatic scaling of plots.
| Number of amplicons | Total number of amplicons listed in primer.bed |
| Number of files | Total number of SAM, BAM or CRAM files |
| End of summary | Always the last item. Marker for end of CSS block. |
This lists summary statistics specific to an individual input file. The values reported are:
| raw total sequences | Total number of sequences found in the file |
| filtered sequences | Number of sequences filtered with -F option |
| failed primer match | Number of sequences that did not correspond to |
| a known primer location | |
| matching sequences | Number of sequences allocated to an amplicon |
For each amplicon, this simply reports the count of reads that have been assigned to it. A read is assigned to an amplicon if the start and/or end of the read is within a specified number of bases of the primer sites listed in the bed file. This distance is controlled via the -m option.
For each amplicon, this lists what percentage of reads were assigned to this amplicon out of the total number of assigned reads. This may be used to diagnose how uniform this distribution is.
Note this is a pure read count and has no relation to amplicon size.
Using the reads assigned to each amplicon and their start / end locations on that reference, computed using the POS and CIGAR fields, we compute the total number of bases aligned to this amplicon and corresponding the average depth. The VDEPTH variants are filtered to only include templates with end-to-end coverage across the amplicon. These can be considered to be "valid" or "usable" templates and give an indication of the minimum depth for the amplicon rather than the average depth.
To compute the depth the length of the amplicon is computed using the innermost set of primers, if multiple choices are listed in the bed file.
Similar to the FDEPTH section, this is a binary status of covered or not covered per position in each amplicon. This is then expressed as a percentage by dividing by the amplicon length, which is computed using the innermost set of primers covering this amplicon.
The minimum depth necessary to constitute a position as being "covered" is specifiable using the -d option.
It is possible for an amplicon to be produced using incorrect primers, giving rise to extra-long amplicons (typically double or treble length).
The FTCOORD field holds a distribution of observed template coordinates from the input data. Each row consists of the file name, the amplicon number in question, and tab separated tuples of start, end, frequency and status (0 for OK, 1 for skipping amplicon, 2 for unknown location). Each template is only counted for one amplicon, so if the read-pairs span amplicons the count will show up in the left-most amplicon covered.
Th COORD data may indicate which primers are being utilised if there are alternates available for a given amplicon.
For COORD lines amplicon number 0 holds the frequency data for data that reads that have not been assigned to any amplicon. That is, they may lie within an amplicon, but they do not start or end at a known primer location. It is not recorded for BED files containing multiple references.
The FAMP / CAMP section is a simple count per amplicon of the number of templates coming from this amplicon. Templates are counted once per amplicon, but and like the FTCOORD field if a read-pair spans amplicons it is only counted in the left-most amplicon. Each line consists of the file name, amplicon number and 3 counts for the number of templates with both ends within this amplicon, the number of templates with the rightmost end in another amplicon, and the number of templates where the other end has failed to be assigned to an amplicon.
Note FAMP / CAMP amplicon number 0 is the summation of data for all amplicons (1 onwards).
These are for depth plots per base rather than per amplicon. They distinguish between all reads in all templates, and only reads in templates considered to be "valid". Such templates have both reads (if paired) matching known primer locations from he same amplicon and have full length coverage across the entire amplicon.
This FDP_VALID can be considered to be the minimum template depth across the amplicon.
The difference between the VALID and ALL plots represents additional data that for some reason may not be suitable for producing a consensus. For example an amplicon that skips a primer, pairing 10_LEFT with 12_RIGHT, will have coverage for the first half of amplicon 10 and the last half of amplicon 12. Counting the number of reads or bases alone in the amplicon does not reveal the potential for non-uniformity of coverage.
The lines start with the type keyword, file / sample name, reference name (unless single-ref mode is enabled), followed by a variable number of tab separated tuples consisting of depth,length. The length field is a basic form of run-length encoding where all depth values within a specified fraction of each other (e.g. >= (1-fract)*midpoint and <= (1+fract)*midpoint) are combined into a single run. This fraction is controlled via the -D option.
This adjustment does not have to be precise as the --pos-margin field permits some leeway. Hence if required, it should be set to approximately double the average primer length.
To run ampliconstats on a directory full of CRAM files and then produce a series of PNG images named "mydata*.png":
samtools ampliconstats V3/nCoV-2019.bed /path/*.cram > astats plot-ampliconstats -size 1200,900 mydata astats
Written by James Bonfield from the Sanger Institute.
samtools(1), samtools-ampliconclip(1) samtools-stats(1), samtools-flags(1)
Samtools website: <http://www.htslib.org/>
| 2 September 2022 | samtools-1.16.1 |