scTE takes as input:
- Aligned sequence reads (BAM/SAM format)
- The genomic location of TEs (BED format)
- The genomic location of genes (GTF format)
scTE works with python >=3.6.
$ git clone https://github.com/hamidghaedi/scTE.git
$ cd scTE
$ python setup.py install
Building genome indices
scTE builds genome indices for the fast alignment of reads to genes and TEs. These indices can be automatically generated using the commands:
$ scTE_build -g mm10 # Mouse
$ scTE_build -g hg38 # Human
$ scTE_build -g panTro6 # Chimpanzee
$ scTE_build -g macFas5 # Macaca fascicularis
$ scTE_build -g dm6 # Drosophila melanogaster
$ scTE_build -g danRer11 # Zebrafish
$ scTE_build -g xenTro9 # Xenopus tropicalis
These scripts will automatically download the genome annotations, for mouse:
$ ftp://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_mouse/release_M21/gencode.vM21.annotation.gtf.gz
$ http://hgdownload.soe.ucsc.edu/goldenPath/mm10/database/rmsk.txt.gz
Or for human:
$ ftp://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_human/release_30/gencode.v30.annotation.gtf.gz
$ http://hgdownload.soe.ucsc.edu/goldenPath/hg38/database/rmsk.txt.gz
Or for Chimpanzee:
$ http://ftp.ensembl.org/pub/release-103/gtf/pan_troglodytes/Pan_troglodytes.Pan_tro_3.0.103.gtf.gz
$ https://hgdownload.soe.ucsc.edu/goldenPath/panTro6/database/rmsk.txt.gz
Or for Macaca fascicularis:
$ http://ftp.ensembl.org/pub/release-102/gtf/macaca_fascicularis/Macaca_fascicularis.Macaca_fascicularis_5.0.102.gtf.gz
$ http://hgdownload.soe.ucsc.edu/goldenPath/macFas5/database/rmsk.txt.gz
Or for Drosophila melanogaster:
$ http://ftp.ensembl.org/pub/release-103/gtf/drosophila_melanogaster/Drosophila_melanogaster.BDGP6.32.103.gtf.gz
$ http://hgdownload.soe.ucsc.edu/goldenPath/dm6/database/rmsk.txt.gz
Or for Zebrafish:
$ http://ftp.ensembl.org/pub/release-103/gtf/danio_rerio/Danio_rerio.GRCz11.103.gtf.gz
$ https://hgdownload.soe.ucsc.edu/goldenPath/danRer11/database/rmsk.txt.gz
Or for Xenopus tropicalis:
$ http://ftp.ensembl.org/pub/release-103/gtf/xenopus_tropicalis/Xenopus_tropicalis.Xenopus_tropicalis_v9.1.103.gtf.gz
$ https://hgdownload.soe.ucsc.edu/goldenPath/xenTro9/database/rmsk.txt.gz
mm10, hg38, panTro6, macFas5, dm6, danRer11, xenTro9
is the genome assembly version.
If you want to use your customs reference, you can use the -gene -te
options:
scTE_build -te TEs.bed -gene Genes.gtf -o custome
-te
Six columns bed file for transposable elements annotation.
-gene
Gtf file for genes annotation.
For more informat about BED and GTF format, see from UCSC.
These annotations are then processed and converted into genome indices. The scTE algorithm will allocate
reads first to gene exons, and then to TEs by default. Hence TEs inside exon/UTR regions of genes annotated
in GENCODE will only contribute to the gene, and not to the TE score. This feature can be changed by
setting –mode/-m inclusive
in scTE, which will instruct scTE to assign the reads to both TEs and genes
if a read comes from a TE inside exon/UTR regions of genes. If you want to remove the TEs inside the intron
of genes, you can sete –mode/-m nointron
in scTE
Analysis of 10x style scRNA-seq data
scTE makes BAM/SAM file as input, highly recommend to use unfiltered alignment file as input.
For bam
file generated by STARsolo etc, the cell barcodes and UMI need to be integrated into the read 'CR:Z' or 'UR:Z' tage as bellow:
$ scTE -i inp.bam -o out -x mm10.exclusive.idx --hdf5 True -CB CR -UMI UR
$ samtools view test.bam
A00269:12:H7YF2DMXX:2 0 chr10 55902580 255 50M * 0 0 GTTCTCTCCGTATGTGAGCATGGGAGATACATCCCAGAAAGGCAGAAGGG FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF NH:i:1 HI:i:1 AS:i:49 nM:i:0 CR:Z:CTAGAGTGTTTCGCTC CY:Z:FFFFFFFFFFFFFFFF UR:Z:TACATGACGC UY:Z:FFFFFFFFFF
A00269:13:H7YF2DMXX:2 0 chr10 55902784 255 50M * 0 0 ATAATCTTTGAGATCTCTGGTGAAAATAAGTAGCATAAAGGACAGAATCA FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF NH:i:1 HI:i:1 AS:i:49 nM:i:0 CR:Z:CTAGAGTGTTTCGCTC CY:Z:FFFFFFFFFFFFFFFF UR:Z:TACATGACGC UY:Z:FFFFFFFFFF
A00269:14:H7YF2DMXX:2 0 chr13 67837311 255 50M * 0 0 CTGTTCATTATTTGAGGAAATCAGGACAGGAAATCAAACATGGCAGAATC FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF NH:i:1 HI:i:1 AS:i:49 nM:i:0 CR:Z:ATCGAGTGTTTCGCTC CY:Z:FFFFFFFFFFFFFFFF UR:Z:TACATGACGC UY:Z:FFFFFFFFFF
A00269:15:H7YF2DMXX:2 0 chr14 114380523 255 50M * 0 0 GATCCAGATTAATTGAGACTGTTGATCCTCCTACAGGGTCGCCCTTCTCC FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF NH:i:1 HI:i:1 AS:i:49 nM:i:0 CR:Z:CTAGAGTGTTTCGCTC CY:Z:FFFFFFFFFFFFFFFF UR:Z:TACATGACGC UY:Z:FFFFFFFFFF
For bam
file generated by Cell Ranger etc, the cell barcodes and UMI need to be integrated into the read 'CB:Z' or 'UB:Z' tage as bellow:
$ scTE -i inp.bam -o out -x mm10.exclusive.idx --hdf5 True -CB CB -UMI UB
$ samtools view test.bam
A00519:758:HTCCHDSXY:3:2535:21296:19774 16 chr1 14021 0 90M * 0 0 TGGATTTCTATCTCCCTGGCTTGGTGCCAGTTCCTCCAAGTCGATGGCACCTCCCTCCCTCTCAACCACTTGAGCAAACTCCAAGACATC ,FFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:F:FFFFFFFFFFFFFFFFFFF:FFFFF NH:i:5 HI:i:1 AS:i:88 nM:i:0 RG:Z:SC3_v3_NextGem_DI_CellPlex_Human_PBMC_10K:0:1:HTCCHDSXY:3 RE:A:I xf:i:0 CR:Z:CTCCCTCCACTGCGAC CY:Z:FFFFFFFFFFFFFFFF CB:Z:CTCCCTCCACTGCGAC-1 UR:Z:AAGGCGTAGTAG UY:Z:FFFFFFFFFFFF UB:Z:AAGGCGTAGTAG
A00519:758:HTCCHDSXY:1:1355:17237:31720 0 chr1 14260 0 90M * 0 0 CTCCCTCTCATCCCAGAGAAACAGGTCAGCTGGGAGCTTCTGCCCCCACTGCCTAGGGACCAACAGGGGCAGGAGGCAGTCACTGACCCC FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF NH:i:5 HI:i:1 AS:i:88 nM:i:0 RG:Z:SC3_v3_NextGem_DI_CellPlex_Human_PBMC_10K:0:1:HTCCHDSXY:1 RE:A:I xf:i:0 CR:Z:TCGTCCACAGTATGAA CY:Z:FFFFFFFFFFFFFFFF CB:Z:TCGTCCACAGTATGAA-1 UR:Z:GACTTATTTTTT UY:Z:FFFFFFFFFFFF UB:Z:GACTTATTTTTT
A00519:758:HTCCHDSXY:3:2227:16703:32080 16 chr1 14411 1 90M * 0 0 TCAGTTCTTTATTGATTGGTGTGCCGTTTTCTCTGGAAGCCTCTTAAGAACACAGTGGCGCAGGCTGGGTGGAGCCGTCCCCCCATGGAG FFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFF:FFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF NH:i:3 HI:i:1 AS:i:88 nM:i:0 RG:Z:SC3_v3_NextGem_DI_CellPlex_Human_PBMC_10K:0:1:HTCCHDSXY:3 RE:A:I xf:i:0 CR:Z:TTGAGTGGTTGTGGCC CY:Z:FFFFFFFFFFFFFFFF CB:Z:TTGAGTGGTTGTGGCC-1 UR:Z:TATAATGCTCAG UY:Z:FFFFFFFFFFFF UB:Z:TATAATGCTCAG
A00519:758:HTCCHDSXY:3:2563:23665:33802 16 chr1 14411 1 90M * 0 0 TCAGTTCTTTATTGATTGGTGTGCCGTTTTCTCTGGAAGCCTCTTAAGAACACAGTGGCGCAGGCTGGGTGGAGCCGTCCCCCCATGGAG FFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF NH:i:3 HI:i:1 AS:i:88 nM:i:0 RG:Z:SC3_v3_NextGem_DI_CellPlex_Human_PBMC_10K:0:1:HTCCHDSXY:3 RE:A:I xf:i:0 CR:Z:TGTTGAGAGGCAATGC CY:Z:FFFFFFFFFFFFFFFF CB:Z:TGTTGAGAGGCAATGC-1 UR:Z:ACGGGTGTGGAG UY:Z:FFFFFFFFFFFF UB:Z:ACGGGTGTGGAG
-i
Input file: BAM/SAM file from CellRanger or STARsolo
-o
Output file prefix
-x
The filename of the index for the reference genome annotation generated by scTE_build
-p
Number of threads to use, Default: 1. scTE takes ~10Gb memory each thread for human and mouse genome.
--hdf5
Save the output as .h5ad formatted file instead of csv file. Default: False
scTE is most tuned to STARsolo or the Cell Ranger pipeline outputs,
and can accept BAM files produced by either of these two programs.
For other aligners, the barcode should be stored in the CR:Z
or CB:Z
tag, and the UMI in the UR:Z
or UB:Z
tag in the BAM file
Analysis of C1 style scRNA-seq data
If the UMI is missing or not used in the scRNA-seq technology (for example on the Fluidigm C1 platform), it can be disabled with –UMI False
(the default is True) switch in scTE. If the barcode is missing it can be disabled with the –CB False
(the default is True),
and instead the cell barcodes will be taken from the names of the BAM files.
$ scTE -i inp.bam -o out -x mm10.exclusive.idx -CB False -UMI False
multiple BAM files can be provided to scTE with the –i
option
$ scTE -i *.bam -o out -x mm10.exclusive.idx -CB False -UMI False
or
$ scTE -i input1.bam,input2.bam,... -o out -x mm10.exclusive.idx -CB False -UMI False
Analysis of scATAC-seq data
The genome indices were prebuilt using:
$ wget -c http://hgdownload.soe.ucsc.edu/goldenPath/mm10/database/rmsk.txt.gz -O mm10.te.txt.gz
$ zcat mm10.te.txt.gz | grep -E 'LINE|SINE|LTR|Retroposon' | cut -f6-8,11 >mm10.te.bed
$ scTEATAC_build -g mm10.te.bed -o mm10.te.atac
Then the bam file can processe using scTE with the command:
scTE_scatacseq -i input.bam -x mm10.te.atac.idx
Citation
If scTE is useful for your research, consider citing Nature Communications (2021)