- Output enhancements:
- Summary.csv statistics output for raw and filtered cells useful for quick run quality assessment.
- --soloCellFilter option for basic filtering of the cells, similar to the methods used by CellRanger 2.2.x.
- Better compatibility with CellRanger 3.x.x:
- --soloUMIfiltering MultiGeneUMI option introduced in CellRanger 3.x.x for filtering UMI collisions between different genes.
- --soloCBmatchWLtype 1MM_multi_pseudocounts option, introduced in CellRanger 3.x.x, which slightly changes the posterior probability calculation for CB with 1 mismatch.
- Velocyto spliced/unspliced/ambiguous quantification:
- --soloFeatures Velocyto option to produce Spliced, Unspliced, and Ambiguous counts similar to the velocyto.py tool developed by LaManno et al. This option is under active development and the results may change in the future versions.
- Support for complex barcodes, e.g. inDrop:
- Complex barcodes in STARsolo with --soloType CB_UMI_Complex, --soloCBmatchWLtype --soloAdapterSequence, --soloAdapterMismatchesNmax, --soloCBposition,--soloUMIposition
- BAM tags:
- CB/UB for corrected CellBarcode/UMI
- GX/GN for gene ID/name
- STARsolo most up-to-date documentation.
The chimeric read alignment reporting has been changed to improve on the specificity of chimeric read detection. Only those chimeric read aligments with alignment scores that exceed the corresponding score of the non-chimeric alignment to the reference genome are now reported. The Chimeric.junction.out file formatting has been updated to include column headers and the alignment scores for both chimeric alignments and the non-chimeric alternative. See the latest STAR manual for full details.
STARsolo is a turnkey solution for analyzing droplet single cell RNA sequencing data (e.g. 10X Genomics Chromium System) built directly into STAR code. STARsolo inputs the raw FASTQ reads files, and performs the following operations
- error correction and demultiplexing of cell barcodes using user-input whitelist
- mapping the reads to the reference genome using the standard STAR spliced read alignment algorithm
- error correction and collapsing (deduplication) of Unique Molecular Identifiers (UMIa)
- quantification of per-cell gene expression by counting the number of reads per gene
STARsolo output is designed to be a drop-in replacement for 10X CellRanger gene quantification output. It follows CellRanger logic for cell barcode whitelisting and UMI deduplication, and produces nearly identical gene counts in the same format. At the same time STARsolo is ~10 times faster than the CellRanger.
The STAR solo algorithm is turned on with:
--soloType Droplet
Presently, the cell barcode whitelist has to be provided with:
--soloCBwhitelist /path/to/cell/barcode/whitelist
The 10X Chromium whitelist file can be found inside the CellRanger distribution, e.g. 10X-whitelist. Please make sure that the whitelist is compatible with the specific version of the 10X chemistry (V1,V2,V3 etc).
Importantly, in the --readFilesIn option, the 1st file has to be cDNA read, and the 2nd file has to be the barcode (cell+UMI) read, i.e.
--readFilesIn cDNAfragmentSequence.fastq.gz CellBarcodeUMIsequence.fastq.gz
Important: the genome index has to be re-generated with the latest 2.7.0x release.
Other parameters that control STARsolo output are listed below. Note that default parameters are compatible with 10X Chromium V2 protocol.
soloCBstart 1
int>0: cell barcode start base
soloCBlen 16
int>0: cell barcode length
soloUMIstart 17
int>0: UMI start base
soloUMIlen 10
int>0: UMI length
soloStrand Forward
string: strandedness of the solo libraries:
Unstranded ... no strand information
Forward ... read strand same as the original RNA molecule
Reverse ... read strand opposite to the original RNA molecule
soloFeatures Gene
string(s) genomic features for which the UMI counts per Cell Barcode are collected
Gene ... genes: reads match the gene transcript
SJ ... splice junctions: reported in SJ.out.tab
soloUMIdedup 1MM_All
string(s) type of UMI deduplication (collapsing) algorithm
1MM_All ... all UMIs with 1 mismatch distance to each other are collapsed (i.e. counted once)
1MM_Directional ... follows the "directional" method from the UMI-tools by Smith, Heger and Sudbery (Genome Research 2017).
1MM_NotCollapsed ... UMIs with 1 mismatch distance to others are not collapsed (i.e. all counted)
soloOutFileNames Solo.out/ genes.tsv barcodes.tsv matrix.mtx matrixSJ.mtx
string(s) file names for STARsolo output
1st word ... file name prefix
2nd word ... barcode sequences
3rd word ... gene IDs and names
4th word ... cell/gene counts matrix
5th word ... cell/splice junction counts matrix
1. Merging and mapping of overlapping paired-end reads.
This feature improves mapping accuracy for paired-end libraries with short insert sizes, where many reads have overlapping mates. Importantly, it allows detection of chimeric junction in the overlap region.
STAR will search for an overlap between mates larger or equal to --peOverlapNbasesMin bases with proportion of mismatches in the overlap area not exceeding --peOverlapMMp .
If the overlap is found, STAR will map merge the mates and attempt to map the resulting (single-end) sequence.
If requested, the chimeric detection will be performed on the merged-mate sequence, thus allowing chimeric detection in the overlap region.
If the score of this alignment higher than the original one, or if a chimeric alignment is found, STAR will report the merged-mate aligment instead of the original one.
In the output, the merged-mate aligment will be converted back to paired-end format.
The developmment of this algorithm was supported by Illumina, Inc.
Many thanks to June Snedecor, Xiao Chen, and Felix Schlesinger for their extensive help in developing this feature.
2. Detection of personal variants overlapping alignments.
Option --varVCFfile /path/to/vcf/file is used to input VCF file with personal variants. Only single nucleotide variants (SNVs) are supported at the moment.
Each variant is expected to have a genotype with two alleles.
To output variants that overlap alignments, vG and vA have to be added to --outSAMattributes list.
SAM attribute vG outputs the genomic coordinate of the variant, allowing for identification of the variant.
SAM attribute vA outputs which allele is detected in the read: 1 or 2 match one of the genotype alleles, 3 - no match to genotype.
3. WASP filtering of allele specific alignments.
This is re-implementation of the original WASP algorithm by Bryce van de Geijn, Graham McVicker, Yoav Gilad & Jonathan K Pritchard. Please cite the original WASP paper: Nature Methods 12, 1061–1063 (2015) .
WASP filtering is activated with --waspOutputMode SAMtag, which will add vW tag to the SAM output:
vW:i:1 means alignment passed WASP filtering, while all other values mean it did not pass.
Many thanks to Bryce van de Geijn for fruitful discussions.
4. Detection of multimapping chimeras.
Previous STAR chimeric detection algorithm only detected uniquely mapping chimeras, which reduced its sensitivity in some cases.
The new algorithm can detect and output multimapping chimeras. Presently, the only output into Chimeric.out.junction is supported.
This algorithm is activated with >0 value in --chimMultimapNmax, which defines the maximum number of chimeric multi-alignments.
The --chimMultimapScoreRange (=1 by default) parameter defines the score range for multi-mapping chimeras below the best chimeric score, similar to the --outFilterMultimapScoreRange parameter for normal alignments.
The --chimNonchimScoreDropMin (=20 by default) defines the threshold triggering chimeric detection: the drop in the best non-chimeric alignment score with respect to the read length has to be smaller than this value.
Many thanks to Brian Haas for testing and feedback.
- --outSAMtlen 1/2 option to select the calculation method for the TLEN field in the SAM/BAM files: 1 ... leftmost base of the (+)strand mate to rightmost base of the (-)mate. (+)sign for the (+)strand mate 2 ... leftmost base of any mate to rightmost base of any mate. (+)sign for the mate with the leftmost base. This is different from 1 for overlapping mates with protruding ends
- --alignInsertionFlush option which defines how to flush ambiguous insertion positions: None: old method, insertions are not flushed; Right: insertions are flushed to the right.
- --outBAMsortingBinsN option to control the number of sorting bins. Increasing this number reduces the amount of RAM required for sorting.
STAR now uses essential c++11 features. Compiling from sources requires gcc 4.7.0 or later.
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It is now possible to add extra sequences to the reference genome ont the fly (without re-generating the genome) by specifying --genomeFastaFiles /path/to/genome/fasta1 /path/to/genome/fasta2 at the mapping stage.
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By default, the order of the multi-mapping alignments for each read is not truly random. The --outMultimapperOrder Random option outputs multiple alignments for each read in random order, and also also randomizes the choice of the primary alignment from the highest scoring alignments. Parameter --runRNGseed can be used to set the random generator seed. With this option, the ordering of multi-mapping alignments of each read, and the choice of the primary alignment will vary from run to run, unless only one thread is used and the seed is kept constant.
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The --outSAMmultNmax parameter limits the number of output alignments (SAM lines) for multimappers. For instance, --outSAMmultNmax 1 will output exactly one SAM line for each mapped read.
New features:
Counting reads per gene while mapping with --quantMode GeneCounts option. A read is counted if it overlaps (1nt or more) one and only one gene. Both ends of the paired-end read are checked for overlaps. The counts coincide with those produced by htseq-count with default parameters.
Requires annotations (GTF or GFF with --sjdbGTFfile option) used at the genome generation step, or at the mapping step.
Outputs read counts per gene into ReadsPerGene.out.tab file with 4 columns which correspond to different strandedness options: column 1: gene ID column 2: counts for unstranded RNA-seq column 3: counts for the 1st read strand aligned with RNA (htseq-count option -s yes) column 4: counts for the 2nd read strand aligned with RNA (htseq-count option -s reverse) Select the output according to the strandedness of your data. Note, that if you have stranded data and choose one of the columns 3 or 4, the other column (4 or 3) will give you the count of antisense reads.
With --quantMode TranscriptomeSAM GeneCounts, and get both the Aligned.toTranscriptome.out.bam and ReadsPerGene.out.tab outputs.
New features:
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The annotations can now be included on the fly at the mapping step, without including them at the genome generation step. At the mapping step, specify --sjdbGTFfile /path/to/ann.gtf and/or --sjdbFileChrStartEnd /path/to/sj.tab, as well as --sjdbOverhang, and any other --sjdb* options. The genome indices can be generated with or without another set of annotations/junctions. In the latter case the new junctions will added to the old ones. STAR will insert the junctions into genome indices on the fly before mapping, which takes 1~2 minutes. The on the fly genome indices can be saved (for reuse) with "--sjdbInsertSave All", into _STARgenome directory inside the current run directory. Default --sjdbOverhang is now set at 100, and does not have to be specified unless you need to change this value.
The "all-sample" 2-pass method can be simplified using this on the fly junction insertion option: (i) run the 1st pass for all samples as usual, with or without annotations (ii) run 2nd pass for all samples, listing SJ.out.tab files from all samples in --sjdbFileChrStartEnd /path/to/sj1.tab /path/to/sj2.tab ...
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New option to activate on the fly "per sample" 2-pass method: "--twopassMode Basic". Default --twopass1readsN is now -1, i.e. using all reads in the 1st pass. 2-pass mode can now be used with annotations, which can be included either at the run-time (see #1), or at the genome generation step. Annotated junctions will be included in both the 1st and 2nd passes.
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Included link (submodule) to Brian Haas' STAR-Fusion code for detecting fusion transcript from STAR chimeric output: https://github.com/STAR-Fusion/STAR-Fusion
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Included Gery Vessere's shared memory implementation for POSIX and SysV. To compile STAR with POSIX shared memory, use
make POSIXSHARED -
New option "--chimOutType WithinBAM" to include chimeric alignments together with normal alignments in the main (sorted or unsorted) BAM file(s). Formatting of chimeric alignments follows the latest SAM/BAM specifications. Thanks to Felix Schlesinger for thorough testing of this option.
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New option "--quantTranscriptomeBan Singleend" allows insertions, deletions ans soft-clips in the transcriptomic alignments, which can be used by some expression quantification software (e.g. eXpress).
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New option "--alignEndsTypeExtension Extend5pOfRead1" to enforce full extension of the 5p of the read1, while all other ends undergo local alignment and may be soft-clipped.