General Options

These options apply globally and modify the behavior of the whole program. They can be placed anywhere in the command line.

-V, --version

Print version number and exit.

-q, --quiet

Suppress all output except fatal error messages.

-v, --verbose

Produce more output, including progress indicators for most operations.

--debug

Produce debugging output in addition to progress information.

-n, --dry-run

Parse command line, optionally print a description of the intended operations, then exit.

--vmem X

Limit virtual memory to X megabytes. If memory runs out, anfo-tool tries to free up memory by forgetting about big files, e.g. genomes. Use this option to avoid swapping or out-of-memory conditions when operations involve big or multiple genomes.

Setting Parameters

A parameter can be set multiple times on the command line and will overwrite previous settings. Any filter option that needs a parameter picks up the last definition that appeared before the filter option.

--set-slope S

Set the slope parameter to S. The slope is used together with the intercept where filters apply to alignment scores; alignments scoring no worse than slope * (length - intercept) are considered good. The default is 7.5.

--set-intercept L

Set the intercept parameter to L. The intercept is used together with the slope where filters apply to alignment scores; alignments scoring no worse than slope * (length - intercept) are considered good. The default is 20.

--set-context C

Set the context parameter to C. The context is the number of surrounding bases of the reference included when printing alignments in text form. The default is 0.

--set-genome G

Set the genome parameter to G. Many filters will only consider the best alignments to this specific genome if it is set. If no genome is set, the globally best alignment is used.

--clear-genome

Clear the genome parameter. Filters apply to the globally best alignment afterwards.

Filter Options

Filters can be applied before merging the inputs or after splitting the back up.

-s, --sort-pos=n

Sort by alignment position while buffering no more than n MiB in memory. If a genome is set, alignments to that genome are used.

-S, --sort-name=n

Sort by read name while buffering no more than n MiB in memory.

-l, --filter-length=L

Retain alignments only for reads of at least L bases length. The reads themselves are kept.

-f, --filter-score

Retain alignments only if their score is good enough. Usesslopeandintercept.

--filter-mapq=Q

Remove alignments with mapping quality below Q.

-h, --filter-hit=SEQ

Keep only reads that have a hit to a sequence named SEQ. If SEQ is empty, reads are kept if they have any hit. If the genome parameter is set, only hits to that genome count.

--delete-hit=SEQ

Delete alignments to SEQ. If SEQ is empty, all alignments are deleted. If the genome parameter is set, only alignments to that genome are deleted.

--filter-qual=Q

Mask out bases with quality below Q. Such a base is replaced by the N ambiguity code.

--multiplicity=N

Keep only reads of molecules that have been sequenced at least N times. Reads are considered to come from the same original molecule if their aligned coordinates are identical.

--subsample=F

Subsample a fraction F of the results. Every read is independently and randomly choosen to be kept or not.

--inside-regions=FILE

Read a list of regions from FILE, then keep only alignments that overlap an annotated region.

--outside-regions=FILE

Read a list of regions from FILE, then keep only alignments that do not overlap an annotated region.

Special Filters

-d, --rmdup=Q

Remove PCR duplicates, clamp quality scores to Q. Two reads are considered to be duplicates, if their aligned coordinates are identical. If a genome is set, the best alignment to that genome is used, else the globally best alignment. Both alignments must be good, as determined by slopeandintercept. For a set of duplicates, a consensus is called, generally increasing the quality scores. If a resulting quality score exceeds Q, it is set to Q. This filter requires the input to be sorted by alignment coordinate on the selected genome.

--duct-tape=NAME Duct-tape overlapping alignments into contigs and call a consensus for them. If a genome is set, alignments to that genome are used, else the globally best alignments. This filter requires input to be sorted by alignment coordinate on the genome. Output is a set of contigs, every position gets assigned a consensus base, a quality score and likelihoods for every possible diallele. (It is called duct-taping because it kind of looks like an assembly, but is not nearly as solid.)

--edit-header=ED

Invoke the editor ED on the text representaion of the stream's header. This can be used to clean up header that have accumulated too much cruft.

Merging Filters

Exactly one merging filter should be given on the command line, all filter options occuring before that are part of the input filter chains, all further filters become output chains. If no merging filter is given, --concat is assumed, and all filters are input filters.

-c, --concat

Concatenate all input streams in the order they appear on the command line.

-m, --merge

Merge sorted input streams, producing a sorted result. All inputs must be sorted in the same way.

-j, --join

Join input streams and retain the single best hits to each genome. Every input stream must contain a record for every read, reads are buffered in memory until all of their hits are collected. This way, joining works well if all inputs are nearly in the same order. If reads are missing from some streams, joining them will waste memory.

--mega-merge

Merge many streams such as those produced by running anfo-sge. Streams that operated on the same reads are joined, then everything is merged.

Output Options

If an output option is given on the command line, the current output filter chain is ended and a new one is started. If no output option is given, a textual representation of the final stream is written to stdout. All output options accept - to write to stdout.

-o, --output FILE

Write native binary stream (a compressed protobuf message) to FILE. Writing a binary stream and reading it back in is lossless.

--output-text FILE

Write protobuf text stream to FILE. If the necessary genomes are available, a textual representation of the alignments is included. If the context parameter is set, that many additional bases of the reference upstream and downstream from the alignment are included.

--output-sam=FILE

Write alignments in SAM format to FILE.

--output-glz FILE

Write contigs in GLZ 0.9 format to FILE. Generating GLZ only works after application of --duct-tape, every contigs becomes a GLZ record.

--output-3aln FILE

Write contigs in a table based format to FILE. The format is still subject to change, see the source code for detailed documentation.

--output-fasta FILE

Write alignments(!) in FastA format to FILE. Alignments are writte as pair of reference and query sequence, aligned coordinates are indicated in the description of the query sequence. If the context parameter is set, that many additional bases of the reference upstream and downstream from the alignment are included. This format is not suggested for any serious use, it exists to support legacy applications.

--output-fastq FILE

Write sequences(!) in FastQ format to FILE. Writing FastQ effectively reconstitutes the input to ANFO if no filtering was done on the results.

--output-table FILE

Write per-alignment statistics to FILE. The file has three colums:Âsequence length, alignment score, difference to next best alignment. It is mainly useful to analyze/visualize the distribution of alignment scores.

--stats FILE

Write simple statistics to FILE. This results in some simple summary statistics of a whole stream: number of aligned sequences, average length, GC content.