A wrapper for the kallisto | bustools workflow for single-cell RNA-seq pre-processing
Fix: Fixed how nascent/mature/ambiguous matrices are stored in anndata/loom files
Fix: The old lamanno workflow (for legacy purposes) should work now.
Anndata/loom files now have nascent/mature layers rather than unspliced/spliced layers.
--workflow=custom can take in multiple FASTA inputs
Allow --d-list to have comma-separated multiple FASTA files with URLs
Command-line options menu cleaned up a bit
Implements all the updates detailed in protocols paper: https://doi.org/10.1101/2023.11.21.568164
ngs-tools>=1.7.3.ref-n has been fully deprecated. (Thanks to @amcdavid for catching a bug)count--workflow kite:10xFB, where bustools project would be called before bustools correct (the order should be opposite). This fix required a bump to the ngs-tools dependency.--workflow lamanno for -x smartseq3.-i has been fully deprecated.-w option) for bulk, smartseq2 and smartseq3 technologies.-x 10XV3_ULTIMA.-n option) will be deprecated in the next major release. It is now recommended to use --include-attribute and --exclude-attribute options, similar to Cellranger's mkref options (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/advanced/references), to kb ref to reduce index size and memory usage.reffasta (genomic FASTA) and/or gtf (gene annotation GTF) arguments. Support from ngs_tools 1.5.13.countSMARTSEQ is now deprecated. All future uses should use BULK, SMARTSEQ2 or SMARTSEQ3.gene_name column (or the adata.var_names if --gene-names is used).--workflow lamanno for -x BULK and -x SMARTSEQ2 technologies.compile command. See below for more information. (#139)v0.48.0.v0.41.0.kb compile is suggested.compilekallisto and/or bustools binary from source. At the most basic level, it downloads the latest release source distributions from the respective GitHub repositories, compiles them, and places them where kb can automatically detect them.target positional argument specifies which binary (or both) to compile. Possible values are kallisto, bustools and all.--url optional argument may be provided with a URL to a remote archive that will be used instead of the latest GitHub release. When this option is used, target may not be all.--ref optional argument may be provided with a commit hash or git tag. When this option is used, target may not be all.-o optional argument may be used to place the compiled binaries in a different directory. Note that if this option is used, --kallisto and --bustools options will have to be set appropriately when running ref or count.--view option may be used to simply view what binaries (their locations and versions) will be used by kb.--remove option may be used to remove existing compiled binaries.--overwrite option may be used to overwrite existing compiled binaries.kallisto compilation follows https://pachterlab.github.io/kallisto/source and has the same dependencies.bustools compilation follows https://bustools.github.io/source and has the same dependencies.--cmake-arguments argument may be used to pass in a string of additional arguments to pass directly to the cmake command. For instance, to manually specify additional include directories, --cmake-arguments "-DCMAKE_CXX_FLAGS='-I /usr/include'"
ref--include-attribute and --exclude-attribute options which can be used to include/exclude specific GTF entries based on their attributes. The argument to these options must be in the form of a key:value pair, where key is a GTF attribute name and value is the value of the aforementioned attribute to include/exclude. Only one of these two options may be specified, and each option may be specified more than once. When multiple --include-attribute are provided, GTF entries that have any one of the attributes will be processed. When multiple --exclude-attribute are provided, GTF entries that have any one of the attributes will not be processed.count--filter-threshold option to specify the barcode filter threshold. This option may only be used when also providing --filter bustools and indicates the minimum number of times a barcode must appear to be retained from filtering. (#142)--strand option to override automatic strandedness setting by kallisto bus. Available options are unstranded, forward, and reverse.transcript_ids column to be a semicolon-delimited string instead of a list (only applicable when --tcc is provided) as a workaround for an issue with writing lists to h5ad with h5py>=3. #141BULK and SMARTSEQ2 technologies. The two technologies behave identically. The FASTQs may be provided either directly via command-line (only for multiplexed samples), in which case kb will perform demultiplexing, or as a single batch definition text file (only for demultiplexed samples). See https://pachterlab.github.io/kallisto/manual section about batch.txt for formatting. This batch textfile may also contain remote urls to FASTQ files, which will be streamed for supported operating systems. Additionally, added --parity, --fragment-l and --fragment-s options, which may only be provided for these technologies. The first must always be provided, indicating the parity of the reads (single, paired), and the latter two may only be provided when --parity single is also provided, specifying the mean length of the fragments and standard deviation of the fragment lengths.SMARTSEQ technology has been deprecated and will be removed in the next release. Instead, SMARTSEQ2 should be used. See previous point for more information.SMARTSEQ3 technology.--dry-run instead of an alias.--umi-gene option, which deduplicates UMIs by gene. Can not be used with smartseq or bulk technologies.--em option, which estimated gene abundances using the EM algorithm. Can not be used with smartseq or bulk technologies, or with --tcc.-o option to bustools count already exists, but as a directory. For instance, counts_unfiltered/cells_x_genes. Such folders are removed before running the command.--gene-names option, which may only be used with --h5ad or -loom and not --tcc. By specifying this option, the output h5ad or loom matrix will be aggregated by gene names instead of IDs.BDWTA (BD Rhapsody), SPLIT-SEQ, Visium (10x).