NAME

samtools ampliconstats - produces statistics from amplicon sequencing alignment file

SYNOPSIS

samtools ampliconstats [options] primers.bed in.sam|in.bam|in.cram...

DESCRIPTION

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.

SS	Amplicon and file counts. Always comes first
AMPLICON	Amplicon primer locations
FSS	File specific: summary stats
FRPERC	File specific: read percentage distribution between amplicons
FDEPTH	File specific: average read depth per amplicon
FVDEPTH	File specific: average read depth per amplicon, full length only
FREADS	File specific: numbers of reads per amplicon
FPCOV	File specific: percent coverage per amplicon
FTCOORD	File specific: template start,end coordinate frequencies per amplicon
FAMP	File specific: amplicon correct / double / treble length counts
FDP_ALL	File specific: template depth per reference base, all templates
FDP_VALID	File specific: template depth per reference base, valid templates only
CSS	Combined summary stats
CRPERC	Combined: read percentage distribution between amplicons
CDEPTH	Combined: average read depth per amplicon
CVDEPTH	Combined: average read depth per amplicon, full length only
CREADS	Combined: numbers of reads per amplicon
CPCOV	Combined: percent coverage per amplicon
CTCOORD	Combined: template coordinates per amplicon
CAMP	Combined: amplicon correct / double / treble length counts
CDP_ALL	Combined: template depth per reference base, all templates
CDP_VALID	Combined: template depth per reference base, valid templates only

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.

SS / AMPLICON

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.

CSS SECTION

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.

FSS SECTION

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

FREADS / CREADS SECTION

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.

FRPERC / CRPERC SECTION

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.

FDEPTH / CDEPTH / FVDEPTH / CVDEPTH SECTION

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.

FPCOV / CPCOV SECTION

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.

FTCOORD / CTCOORD / FAMP / CAMP SECTION

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).

FDP_ALL / CDP_ALL / FDP_VALID / CDP_VALID section

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.

OPTIONS

-f, --required-flag INT|STR

Only output alignments with all bits set in INT present in the FLAG field. INT can be specified in hex by beginning with `0x' (i.e. /^0x[0-9A-F]+/) or in octal by beginning with `0' (i.e. /^0[0-7]+/) [0], or in string form by specifying a comma-separated list of keywords as listed by the "samtools flags" subcommand.

-F, --filter-flag INT|STR

Do not output alignments with any bits set in INT present in the FLAG field. INT can be specified in hex by beginning with `0x' (i.e. /^0x[0-9A-F]+/) or in octal by beginning with `0' (i.e. /^0[0-7]+/) [0], or in string form by specifying a comma-separated list of keywords as listed by the "samtools flags" subcommand.

-a, --max-amplicons INT

Specify the maximum number of amplicons permitted.

-b, --tcoord-bin INT

Bin the template start,end positions into multiples of NT prior to counting their frequency and reporting in the FTCOORD / CTCOORD lines. This may be useful for technologies with higher errors rates where the alignment ends will vary slightly. Defaults to 1, which is equivalent to no binning.

-c, --tcoord-min-count INT

In the FTCOORD and CTCOORD lines, only record template start,end coordinate combination if they occur at least INT times.

-d, --min-depth INT

Specifies the minimum base depth to consider a reference position to be covered, for purposes of the FRPERC and CRPERC sections.

-D, --depth-bin FRACTION

Controls the merging of neighbouring similar depths for the FDP_ALL and FDP_VALID plots. The default FRACTION is 0.01, meaning depths within +/- 1% of a mid point will be aggregated together as a run of the same value. This merging is useful to reduce the file size. Use -D 0 to record every depth.

-l, --max-amplicon-length INT

Specifies the maximum length of any individual amplicon.

-m, --pos-margin INT

Reads are compared against the primer start and end locations specified in the BED file. An aligned sequence should start precisely at these locations, but sequencing errors may cause the primer clipping to be a few bases out or for the alignment to add a few extra bases of soft clip. This option specifies the margin of error permitted when matching a read to an amplicon number.

-o FILE

Output stats to FILE. The default is to write to stdout.

-s, --use-sample-name

Instead of using the basename component of the input path names, use the SM field from the first @RG header line.

-S, --single-ref

Force the output format to match the older single-reference style used in Samtools 1.12 and earlier. This removes the reference names from the SS, AMPLICON, DP_ALL and DP_VALID sections. It cannot be enabled if the input BED file has more than one reference present. Note that plot-ampliconstats can process both output styles.

-t, --tlen-adjust INT

Adjust the TLEN field by +/- INT to compensate for primer clipping. This defaults to zero, but if the primers have been clipped and the TLEN field has not been updated using samtools fixmate then the template length will be wrong by the sum of the forward and reverse primer lengths.

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.

-@ INT

Number of BAM/CRAM (de)compression threads to use in addition to main thread [0].

EXAMPLE

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

AUTHOR

Written by James Bonfield from the Sanger Institute.