BioBloom Tools User Manual
BioBloom Tools (BBT) provides the means to create filters for a given reference and then to categorize sequences. This methodology is faster than alignment but does not provide mapping locations. BBT was initially intended to be used for pre-processing and QC applications like contamination detection, but is flexible to accommodate other purposes. This tool is intended to be a pipeline component to replace costly alignment steps.
Multi-index Bloom filters Manual - See here if classifying to many (>100 targets) different references at the same time
BioBloom tools: fast, accurate and memory-efficient host species sequence screening using bloom filters.
Justin Chu, Sara Sadeghi, Anthony Raymond, Shaun D. Jackman, Ka Ming Nip, Richard Mar, Hamid Mohamadi, Yaron S. Butterfield, A. Gordon Robertson, Inanç Birol
Bioinformatics 2014; 30 (23): 3402-3404.
This tool is free for academic use (GPL3 licence).
We have a commercial licence also, please contact us (prebstein at bccancer dot bc dot ca) if you wish to use the tools for commercial uses.
Any questions, comments and suggestions can be directed to @JustinChu or emailed to email@example.com.
Canada’s Michael Smith Genome Sciences Centre, BC Cancer Agency, Vancouver BC Canada V5Z 4S6
Department of Bioinformatics, University of British Columbia, Vancouver BC V6T 1Z4
Table of Contents
- Compiling and Installing BioBloomTools
- How to Install
- Generating Bloom Filters from Reference Sequence with Biobloommaker
- Classifying and Analyzing Sequences with Biobloomcategorizer
- Program Outputs
- Bloom Filter File (filterID.bf)
- Bloom Filter Info File (filterID.txt)
- Summary File (summary.tsv)
- Categorized Sequence FastA/FastQ Files
- Understanding BioBloomTools
- About Bloom Filters
- How false positive rates correlates to memory usage
- How many hash functions should be used?
- K-mer Tiling and Reduction of the effect of false positive k-mers
- What is inside the Bloom Filter Info File?
- Obtaining the number of unique k-mers in the reference
- Obtaining the number of redundant k-mers in the reference
- Specifications on memory, cpu and storage requirements
- Advanced options and Best Practices
1. Compiling and Installing BioBloomTools
- Autotools (if directly cloning from repo)
- Google Sparsehash
If cloning directly from the repository make sure you get the required submodules:
git submodule update --init
If cloning directly from the repository run:
Compiling BioBloomTools should be as easy as:
./configure && make
To install BBT in a specified directory:
./configure --prefix=/BBT/PATH && make install
If your boost library headers are not in your PATH you can specify their location:
./configure –-with-boost=/boost/path --prefix=/BBT/PATH && make install
BioBloomTools can be installed on linux using conda:
conda install -c bioconda -c conda-forge biobloomtools
Alternatively, if you have linuxbrew/homebrew you can run:
brew install biobloomtools
2. Generating Bloom Filters from Reference Sequences with Biobloommaker
After you have your FastA file a
.bf file with corresponding information text file can be created by running the command:
./biobloommaker –p input input1.fasta input2.fasta
-p is the prefix for the files being created, it also acts as an ID for the filter.
The options above are the bare minimum options you must use to run the program, but it is possible to customize many aspects of your filter that can drastically change performance depending on your needs. See section 5 for advanced options. You can also use the
-h command for a listing of the options.
The optimal size of the filter will be calculated based on the maximum false positive rate (default is 0.075) and the number of hash functions (can be set but is optimized based on FPR).
Two files will be generated binary Bloom filter file (
.bf) and an information file in INI format (
.txt). The information file must be kept with the
.bf file to provide all the needed information to run the categorization.
3. Classifying and Analyzing Sequences with Biobloomcategorizer
Important Note: Bloom filters in versions of bbt where the second number of the version is incremented, will not be compatible, and will result in undefined/meaningless behavior. For example a filter created in 2.0.12 will work with 2.0.13 but not 2.1.X or 2.2.X etc. We do not produce an error message at present regarding this prior to 2.3.0.
Once you have filters created, you can use them with Biobloomcategorizer to categorize sequences. The file formats that can be used are the following: FastQ, FastA. If the input is compressed it is recommended to pipe the input in using process subsititution.
Before starting make sure the listed
.bf file is in the same directory as its corresponding information
readStatus.tsv summary file, and FastA files containing categorized reads can be generated with the following command:
./biobloomcategorizer –-fa –p /output/prefix –f "filter1.bf filter2.bf filter3.bf" inputReads1.bam.bz2 inputreads2_qseq.txt
-p is the output directory for the output files.
-f is the filter(s) you used to categorize the sequences. You can specify as many as
There are some advanced options open can use outlined in section 5. Notable option
one can use is the paired end mode
./biobloomcategorizer -e –p /output/prefix –f "filter1.bf filter2.bf filter3.bf" inputReads1_1.fq inputreads1_2.fq
-e will require that both reads match when making the call about what reference they belong in.
-e will only count a read if both reads match a filter. If you want only it to count situations where only one read matches the filter then the
--inclusive) option can also be used.
These are general use cases you can use to run the program, but it is possible to customize many aspects of your filter that can drastically change performance depending on your needs. See section 6 for advanced options. You can also using the
-h command for a listing on the options.
4. Program Output
i. Bloom Filter File (
- Simply a bit array representing the bloom filter dumped as a file. It is simple, so that it can be used in almost any system format. It is useless without its paired info file however.
ii. Bloom Filter Info File (
- This is the information file of the bloom filter, containing the information like false positive rate and hash functions used. It is in human readable INI format. It is intended to be read by Biobloomcategorizer in tandem with its paired
.bffile to perform categorization.
i. Summary File (
- Tab separated file. Contains proportion information about reads mapping to each filter. Gives a overview of your results.
hitscolumn is the number of hits to this filter, regardless if unique.
sharedcolumn a subset of hits that also hit other filter.
multiMatchentry refers to any hits are shared at least 2 genomes.
noMatchentry is a subset of reads that do not match any of the filters used.
ii. Categorized Sequence FastA/FastQ Files
- In the output directory there will be files for every filter used in addition to
noMatchfiles. The reads will be categorized in these locations based on the threshold
- Reads outputted will have a value (e.g.
/1) appended to the end of each ID to denote pair information about the read.
5. Understanding BioBloomTools
A. About Bloom Filters
The whole idea of using bloom filter centers on getting the time complexity of a hash (i.e. a
O(1) time complexity for look-ups) with a lower space requirement. This is resolved in a bloom filter by not storing the entry, but rather storing the entry’s bit signature (determined via hashing) in a bit array. However, this means there is no collision detection and all bloom filters will have some sort of false positive rate associated with them. The false positive rate must be carefully considered as it determines the expected size of the filter used. Too small of a false positive rate can mean a large bloom filter, but too large of a false positive rate could introduce too much error for the filter to be practical.
B. How false positive rates correlates to memory usage
This figure shows the relationship (assuming optimal number of hash functions have been used) between false positive rate and the space cost per entry. To use this chart divide your amount of space in bits you have to work with to the number base pairs you have in your reference.
For example, say I want the human genome (~
3.4×10^9) filter to fit into~3GB of memory.
(8×3×230)/4×10^9 = ~8bits per entry, meaning a filter with 2% FPR at maximum should be used.
C. How many hash functions should be used?
The number of hash functions refers to the number of hash functions used by a single filter per element. In practice the approximate optimal number of hash functions will be calculated automatically by our program.
We give users the ability to change the number of hash functions because very low false positive rates will have an optimal number of associated hash functions that may be very large and may slow down classification. Also it is recommended that if running multiple filters at the same time that they all use the same number of hash functions.
D. K-mer Tiling
We use a sliding window across each read of size k to categorize sequences. Single base overlaps that both hit a filter are unlikely to be false positives. This information is used to reduce the effect of any false positives. This concept is also used to further improve speed, we also employ a jumping k-mer (rather than sliding) heuristic that skips k k-mers when a miss is detected after a long series of adjacent hits.
E. What is inside the Bloom Filter Info File?
i. Obtaining the number of unique k-mers in the reference:
Within the information txt file for each bloom filter there is a "num_entries" entry that lets you know how many unique k-mers have been added to the filter. It is a lower bound estimate due to possible false positives.
ii. Obtaining the number of redundant k-mers in the reference:
Within the information txt file for each bloom filter there is a
redundant_sequences entry that lets you know how many redundant k-mers have been added to the filter. It is an upper bound estimate due to possible false positives. The
redundant_fpr represents the probability a unique entry could be mis-classified as redundant. Thus, to get the approximate number of unique k-mers take the
redundant_fpr value and multiply it with the
num_entries and add that to the
Expected total number of k-mers = (
F. Specifications on memory, cpu and storage requirements
Memory usage is determined by the size of the database given so there is no "optimal" memory requirements. There is a bit of overhead but the formula roughly matches this:
m=(-ln(p)/((ln(2))^2)) * n where
m is size of the filter in bits,
p is the FPR and
n is the number of elements (number of bases in reference fasta file).
If used in an job based automated cluster environment either the user can specify memory of you can infer memory usage based on the input bloom filter sizes (e.g. size of the input bf files + 100mb overhead).
When creating filters: Again, as alluded to above, storage is dependant of the input reference fasta file, following the same formula. Here is a nice sanity check: memory usage is roughly equal the sum of sizes of the raw bloom filter file used and vice versa.
When filtering reads:
The size of the output is proportional to the input since the results need to be stored. The contents of the output fastq files can be compressed directly as needed with the
--gz option however (which is what
zlib was needed in the installation).
BBT does not create temporary files so scratch space is not needed.
If used in an job based automated cluster environment where users have their own allocated storage they should make sure they have space for the output bloom filter. When categorizing reads they should make sure they have space for the output (if they want the reads
--fq) which will be roughly the size of the input files since all they are doing is partitioning the reads the reads.
There is no cpu minimum speed or number of cores, though it will run faster with more and faster cpus. In terms of a maximum, speed can become I/O bound quickly. When using only a few bloom filters(<5) in BBC the number of cores (>4) may not matter too much, but you will get better performance with multiple threads if more bloom filters are used at the same time.
6. Advanced options and Best Practices
Help dialog from the
--help options has the most up to date information about all the options in this tool and consulting it should give you good information about the effects of some options.
Usage: biobloommaker -p [FILTERID] [OPTION]... [FILE]... Usage: biobloommaker -p [FILTERID] -r 0.2 [FILE]... [FASTQ1] [FASTQ2] Creates a bf and txt file from a list of fasta files. The input sequences are cut into a k-mers with a sliding window and their hash signatures are inserted into a bloom filter. -p, --file_prefix=N Filter prefix and filter ID. Required option. -o, --output_dir=N Output location of the filter and filter info files. -h, --help Display this dialog. --version Display version information. -v --verbose Display verbose output. -t, --threads=N The number of threads to use. Bloom filter options: -f, --fal_pos_rate=N Maximum false positive rate to use in filter. [0.0075] -g, --hash_num=N Set number of hash functions to use in filter instead of automatically using calculated optimal number of functions. -k, --kmer_size=N K-mer size to use to create filter.  -d, --no_rep_kmer Remove all repeat k-mers from the resulting filter in progressive mode. -n, --num_ele=N Set the number of expected elements. If set to 0 number is determined from sequences sizes within files.  Options for progressive filters: -r, --progressive=N Progressive filter creation. The score threshold is specified by N, which may be either a floating point score between 0 and 1 or a positive integer. If N is a positive integer, it is interpreted as the minimum number of contiguous matching bases required for a match. -s, --subtract=N Path to filter that you want to uses to minimize repeat propagation of k-mers inserted into new filter. You may only use filters with k-mer sizes equal the one you wish to create. -d, --no_rep_kmer Remove all repeat k-mers from the resulting filter in progressive mode. -a, --streak=N The number of hits tiling in second pass needed to jump Several tiles upon a miss. Progressive mode only.  -l, --file_list=N A file of list of file pairs to run in parallel. -b, --baitScore=N Score threshold when considering only bait. [r] -e, --iterations=N Pass through files N times if threshold is not met. -i, --inclusive If one paired read matches, both reads will be included in the filter. Only active with the (-r) option. -I, --interval the interval to report file processing status  -P, --print_reads During progressive filter creation, print tagged reads to STDOUT in FASTQ format for debugging [disabled] Report bugs to <firstname.lastname@example.org>.
Usage: biobloomcategorizer [OPTION]... -f "[FILTER1]..." [FILE]... biobloomcategorizer [OPTION]... -e -f "[FILTER1]..." [FILE1.fq] [FILE2.fq] biobloomcategorizer [OPTION]... -e -f "[FILTER1]..." [SMARTFILE.fq] Categorize Sequences. The input format may be FASTA, FASTQ, and compressed gz. -p, --prefix=N Output prefix to use. Otherwise will output to current directory. -f, --filter_files=N List of filter files to use. Required option. eg. "filter1.bf filter2.bf" -e, --paired_mode Uses paired-end information. For BAM or SAM files, if they are poorly ordered, the memory usage will be much larger than normal. Sorting by read name may be needed. -i, --inclusive If one paired read matches, both reads will be included in the filter. -s, --score=N Score threshold for matching. N may be either a floating point score between 0 and 1 or a positive integer representing the minimum match length in bases. If N is a floating point, the maximum threshold is any number less than 1, and the minimum is 0 (highest sensitivity). If set to 1, the best hit is used rather than the threshold and the score will be appended to the header of the output read. [0.15] -w, --with_score Output multimatches with scores in the order of filter. -t, --threads=N The number of threads to use.  -g, --gz_output Outputs all output files in compressed gzip. --fa Output categorized reads in Fasta files. --fq Output categorized reads in Fastq files. --chastity Discard and do not evaluate unchaste reads. --no-chastity Do not discard unchaste reads. [default] -l, --file_list=N A file of list of file pairs to run in parallel. -v, --version Display version information. -h, --help Display this dialog. --verbose Display verbose output -I, --interval the interval to report file processing status  Advanced options: -r, --streak=N The number of hits tiling in second pass needed to jump Several tiles upon a miss. Small values decrease runtime but decrease sensitivity.  -c, --ordered Use ordered filtering. Order of filters matters (filters listed first have higher priority). Only taken advantage of when k-mer sizes and number of hash functions are the same. -d, --stdout_filter Outputs all matching reads to stdout for the first filter listed by -f. Reads are outputed in fastq, and if paired will output will be interlaced. -n, --inverse Inverts the output of -d (everything but first filter). Report bugs to <email@example.com>.
A. How can distinguish between organisms that share lots of k-mer content?
-w) in biobloomcategorizer will output the score of each filter (in the order specified with
-f) in the header of the multimatch filter.
Using this option will make the program run a bit slower but will allow users to see the scores assigned to each filter, so as to make a more informed decision about how the read should be binned.
B. How can I reduce my memory usage?
Memory usage is directly dependent on the filter size, which is in turn a function of the false positive rate. In biobloommaker reducing memory increases the false positive rate (
-f) until the memory usage is acceptable. You may need to increase score threshold (
-s) in biobloomcategorizer to keep the specificity high.
C. How can I make my results more sensitive?
In biobloomcategorizer try to decrease the score threshold (
-s). If that still does not work, in biobloommaker try reducing the k-mer (
-k) size to allow more tiles, which can help with sensitivity. Note that if
-k is decreased to values that cause sequence collisions, this can cause non-specific classification (for most tasks
k=16 is probably as low as you can go).
D. How can I make my results more specific?
In biobloomcategorizer you can increase score threshold (
In biobloommaker decreasing the false positive rate (
-f) can help with specificity. Decreasing the filter false positive rate will increase memory usage.
Due to seqeunce collisions, increasing
-k can improve the effective specificity because they can be used to obtain more unique sequence matches (higher k has higher complexity). There will be fewer frames to consider when increasing the k-mer which may slightly increase the chance of random false positives.
E. How can I make the program faster?
There are multiple ways to speed up biobloomcategorizer. Here are a few options:
--ordered option, other than priotizing the first filters in the list (specified by
-f), will have an added benefit of speeding up the program by avoiding some evaluations if a match is already found. Furthermore, because of this speed up, this option maybe appropriate even in situations where no hierarchy is desired (filters must be unrelated in this case).