nf for axolotl visium processing with kallisto

The original codes were from Tomas and Ashley from Barbara group ETH and highly appreciate that they were willing to share.

Documentation of nf visium initially by Tomas.

IMPORTANT:

in the kallisto_pipeline.nf file, there is a command for running spaceranger (you can Ctrl+F "spaceranger" to find it). you will need to edit the path for the mock fastq reads to include where you're saving those that I'm sending. For example the current path there is "/links/groups/treutlein/USERS/tomasgomes/gene_refs/human/refdata-gex-GRCh38-2020-A/mock_fastq", this will need to be changed to the folder now containing the "mock_fastq" folder I'm sending.

Example of a command to quantify Visium data with the Axolotl transcriptome:

nextflow run kallisto_pipeline.nf
	--transcriptome /links/groups/treutlein/USERS/tomasgomes/gene_refs/axolotl/Amex_T_v47/cDNA_transcripts/AmexT_v47_artificial.fa
	--transindex AmexT_v47_artificial.kalid
	--t2g /links/groups/treutlein/USERS/tomasgomes/gene_refs/axolotl/Amex_T_v47/cDNA_transcripts/AmexT_v47_artificial_genenames_t2g.txt
	--white /links/groups/treutlein/USERS/tomasgomes/gene_refs/other/visium-v1_whitelist_kallisto.txt
	--samplename "A1_limb"
	--outdir /links/groups/treutlein/USERS/tomasgomes/data/axolotl/
	--protocol visiumv1
	--reads "/links/groups/treutlein/DATA/sequencing/20200821_P1288_ASHLEY_VISIUM_axolotl_visium_control_11dpa/raw/A1_Animal1_Control/*.fastq.gz"
	--images "V19S23-109"
	--imagear "A1"
	--imagef "/links/groups/treutlein/DATA/sequencing/20200821_P1288_ASHLEY_VISIUM_axolotl_visium_control_11dpa/image/A1_large_image1.jpg"
	--imageal "/links/groups/treutlein/DATA/sequencing/20200821_P1288_ASHLEY_VISIUM_axolotl_visium_control_11dpa/alignment_files/V19S23-109-A1.json"

Command breakdown:

nextflow run kallisto_pipeline.nf

# this is the fasta transcriptome of axolotl. in this case it also includes some sequences for eGFP etc, although that might not be needed.
# the fasta headers just have the isoform name (e.g. ">AMEX60DD301000001.2")
# I originally got this from Sergej, but can also be obtained from the axolotl website
--transcriptome /links/groups/treutlein/USERS/tomasgomes/gene_refs/axolotl/Amex_T_v47/cDNA_transcripts/AmexT_v47_artificial.fa

# this is the indexed transcriptome for kallisto. if it doesn't exist, it'll be generated from the fasta file, and saved in the same folder
# if it does exist, the fasta file still has to be specified, and they have to be in the same folder
--transindex AmexT_v47_artificial.kalid

# this is a table with the isoform name in one column and the gene in another column. it's required to summarise the kallisto results by gene
# can use either the gene name or gene ID, however you prefer to summarise the data
# example row: AMEX60DD301000001.1	ZNF568
--t2g /links/groups/treutlein/USERS/tomasgomes/gene_refs/axolotl/Amex_T_v47/cDNA_transcripts/AmexT_v47_artificial_genenames_t2g.txt

# this is the whitelist for the visium barcodes (present in spaceranger, also needed for kallisto)
--white /links/groups/treutlein/USERS/tomasgomes/gene_refs/other/visium-v1_whitelist_kallisto.txt

# this is the name given to the sample
--samplename "A1_limb"

# the directory where to output the results
--outdir /links/groups/treutlein/USERS/tomasgomes/data/axolotl/

# the protocol being used (since the pipeline also works for other types of data)
--protocol visiumv1

# the path for the reads. this has to be passed like in this example (i.e. all fastq files in the same folder,
# containing either R1 or R2 in the name, just passed with the "*" to automatically list all of them)
# example file names: A1_Animal1_Control_S1_L001_R1_001.fastq.gz and A1_Animal1_Control_S1_L001_R2_001.fastq.gz
--reads "/links/groups/treutlein/DATA/sequencing/20200821_P1288_ASHLEY_VISIUM_axolotl_visium_control_11dpa/raw/A1_Animal1_Control/*.fastq.gz"

# these next parameters are all for spaceranger
# Visium slide serial number, for example 'V10J25-015'
--images "V19S23-109"

# Visium area identifier, for example 'A1'
--imagear "A1"

# path to the H&E brightfield image file
--imagef "/links/groups/treutlein/DATA/sequencing/20200821_P1288_ASHLEY_VISIUM_axolotl_visium_control_11dpa/image/A1_large_image1.jpg"

# Alignment file produced by the manual Loupe alignment step
# this is something that Ashley did (manually aligning the images and the spots in Loupe), if you need help you can ask her
--imageal "/links/groups/treutlein/DATA/sequencing/20200821_P1288_ASHLEY_VISIUM_axolotl_visium_control_11dpa/alignment_files/V19S23-109-A1.json"

my own questions:

general
- this is not only a nf pipeline for visium, but also for plate-based or 10x scRNA-seq, but all kallisto-based ?
input of pipeline
- whitelist input is only one column of barcode ?
- protocol visiumv1 ?
- imageal ?
output of pipeline
- genecounts.barcodes.txt is UMI count right ?
- UMI rank was plotted, but filter was not done (not for visium)?
- umi duplication plots, what do two plots mean ? and also the four columns of umicount.txt
- estimateAmbience ? (control the cross-contamination from the environment)
analysis UMI counts from kallisto (similar to other count table ?)
- SCT transform (Tomas uses SCT and Ashely scaling factor-based)
- cell type deconvolution (tey use cell2locate)

lengfei5/nf_visium_kallisto

nf for axolotl visium processing with kallisto

Documentation of nf visium initially by Tomas.

my own questions: