Preprocessing transcriptomic library¶
After sequencing, we proceed with the preprocessing of the data, to go from raw reads to transcriptomic information mapped to the mouse genome, in space.
Demultiplexing¶
We got basecall files in bcl format from our sequencing facility.
We used bcl2fastq for demultiplexing, using this sample sheet.
We used the conda environment where we installed spacemake (see instructions
on how to install spacemake), and ran the following commands:
(base) user@computer:~$ bcl2fastq \
--no-lane-splitting \
--runfolder-dir /openst/data/0_basecalls/230616_VH01346_22_AAC2LVVHV \
-o /openst/data/1_spacemake_mouse/demultiplexed_data \
--sample-sheet /openst/data/0_sample_sheets/230616_NR_FC_ST_72_76_AT_01.csv
We obtained fastq files that will be used for the rest of the pipeline, for Read 1 and Read 2. As well, this library was resequenced (add more depth to the data); these resequenced Read 1 and resequenced Read 2 files are available.
Once you download these files, you can move them anywhere in your filesystem. We assume that you have opened a terminal,
and you have browsed to your home directory. From there, create a folder openst_e13_mouse_head_demo; browse inside, and create
another folder data. Then, copy the folder with the fastq files in here. You should have a structure like:
Transcriptomic & spatial mapping with spacemake¶
First of all, intialize the conda environment for spacemake
Initialize¶
Create two folders inside your openst_e13_mouse_head_demo folder, called spacemake and bins, so you will have:
Download the DropSeq tools,
decompress it, and put it inside the bins subdirectory.
Then, following the spacemake Quick start guide,
browse to the spacemake directory you just created in the openst_e13_mouse_head_demo folder, and run the initialization:
(spacemake) user@computer:~$ cd /home/user/openst_e13_mouse_head_demo/spacemake
(spacemake) user@computer:/home/user/openst_e13_mouse_head_demo/spacemake$ spacemake init
--dropseq_tools /home/user/bins/Drop-seq_tools-2.5.1
Configure¶
As spacemake comes with no default value for species, before anything can be done, a new species has to be added.
In this case, we add mouse; you will need to download the correct fa and gtf files. For instance, you can download the
mouse genome from gencode,
as well as the annotation.
Then, you need to run the following commands (remember, in the same spacemake folder as before, with the spacemake conda environment;
we are going to omit (spacemake) user@computer:/home/user/openst_e13_mouse_head_demo/spacemake$ for brevity).
spacemake config add_species \
--name mouse \
--reference genome \
--sequence GRCm39vM30.genome.fa \
--annotation gencodevM30.annotation.gtf
spacemake config add_species \
--name mouse \
--reference rRNA \
--sequence mouse.rRNA.fa
spacemake config add_species \
--name mouse \
--reference phiX \
--sequence phiX.fa
Note
The .fa and .gtf files for mouse are available for http download under the example datasets page.
For instance, you can run:
Add sample¶
Now you need to add the sample data and metadata to spacemake. For this, you will also need the puck (tile) barcode files, which can be
generated with the openst package.
For simplicity, we provide the tile barcode files that are related to this sample, as well as the coordinate system for the Illumina flow cell that was used to generate the capture area of this experiment.
When downloading the tile barcode files, create a folder under openst_e13_mouse_head_demo/data called tiles. Move the files of the tile barcode files
into this folder. Also, move the coordinate file to the puck_data folder in the spacemake directory.
Remember! You need to be in the /home/user/openst_e13_mouse_head_demo/spacemake directory (or similar, depending on what you created);
then run the following commands:
# downloading R1 and R2 sequences
wget "http://bimsbstatic.mdc-berlin.de/rajewsky/openst-public-data/e13_mouse_head_R1_001.fastq.gz"
wget "http://bimsbstatic.mdc-berlin.de/rajewsky/openst-public-data/e13_mouse_head_R2_001.fastq.gz"
wget "http://bimsbstatic.mdc-berlin.de/rajewsky/openst-public-data/e13_mouse_head_reseq_R1_001.fastq.gz"
wget "http://bimsbstatic.mdc-berlin.de/rajewsky/openst-public-data/e13_mouse_head_reseq_R2_001.fastq.gz"
# downloading the spatial barcode sequences
wget "http://bimsbstatic.mdc-berlin.de/rajewsky/openst-public-data/e13_mouse_head_tiles.tar.xz"
tar -xvf e13_mouse_head_tiles.tar.xz
spacemake projects add_sample \
--project_id openst_demo \
--sample_id openst_demo_e13_mouse_head \
--R1 e13_mouse_head_R1_001.fastq.gz e13_mouse_head_reseq_R1_001.fastq.gz \
--R2 e13_mouse_head_R2_001.fastq.gz e13_mouse_head_reseq_R2_001.fastq.gz \
--species mouse \
--puck openst \
--run_mode openst \
--barcode_flavor openst \
--puck_barcode_file e13_mouse_head_tiles/*.txt.gz \
--map_strategy "bowtie2:phiX->bowtie2:rRNA->STAR:genome:final"
You can specify the coordinate system by modifying the openst run mode in the config.yaml file that is created
after you run the spacemake init command (see above). Modify the following lines from this:
into this:
openst:
coordinate_system: puck_data/fc_1_coordinate_system.csv
spot_diameter_um: 0.6
width_um: 1200
You can download the coordinate system file from the Open-ST website, for example:
# download the coordinate system
wget "http://bimsbstatic.mdc-berlin.de/rajewsky/openst-public-data/fc_1_coordinate_system.csv"
cp fc_1_coordinate_system.csv puck_data/.
Run¶
That's all you need to configure! Now, you can run spacemake with the following:
You can modify the number of --cores depending on your local machine; also, you can specify additional
arguments to spacemake run - refer to the official documentation.
Expected output¶
Once spacemake finishes, you will see that several folders and files have been created under projects
(inside the spacemake directory). For example, you can check the QC reports in your web browser by opening the
file at projects/openst_demo/processed_data/openst_demo_e13_mouse_head/illumina/complete_data/qc_sheets/qc_sheet_openst_demo_e13_mouse_head_fc_1_puck_collection.html,
giving you a first impression of what's the quality of spatial mapping, amount of transcripts and genes per barcoded spot, and others.
Importantly, you will find files in the directory projects/openst_demo/processed_data/openst_demo_e13_mouse_head/illumina/complete_data/dge
with the name dge.all.polyA_adapter_trimmed.mm_included.spatial_beads_*.h5ad (where * is a wildcard). These files, and not the ones containing
the words mesh, hexagon or circle are the ones that will be used later to perform the pairwise alignment with imaging data, and to
later reconstruct the cell-by-gene matrix.
If you specified options for meshing in the run_mode, there will be a file containing keywords puck_collection and mesh, hexagon or circle.
This contains approximate cell-by-gene information, as the transcripts are aggregated by a regular lattice and not by the true spatial arrangement of
cells. This might be already enough for some analyses.
Anyway... keep going with the tutorial if you want to unleash the full potential of Open-ST.