10x Genomics Single Cell 3’ V3#

Check this GitHub page to see how 10x Genomics Single Cell 3’ V3 libraries are generated experimentally. This is a droplet-based method, where cells are captured inside droplets. At the same time, gel beads with barcoded oligo-dT primer containing UMIs are also captured inside the droplet. Reverse transcription happens inside the droplet. The cells and gel beads are loaded on the microfluidic device at certain concentrations, such that a fraction of droplets contain only one cell AND one bead.

The V3 chemistry is a significant improvement over the previous V2 version of the kit in terms of sensitivity: many more detected genes per cell. The library structure is extremely similar to the V2 version.

For Your Own Experiments#

Your sequencing read configuration is like this:

Order	Read	Cycle	Description
1	Read 1	28	This yields `R1_001.fastq.gz`, 16 bp cell barcodes + 12 bp UMI
2	Index 1 (i7)	8 or 10	This yields `I1_001.fastq.gz`, Sample index
3	Index 2 (i5)	8 or 10 or None	This yields `I2_001.fastq.gz`, Sample index (if using dual index)
4	Read 2	>50	This yields `R2_001.fastq.gz`, cDNA reads

If you sequence your data via your core facility or a company, you will need to provide the sample index sequence, which is the primer (PN-1000213/PN-2000240) taken from the commercial kit from 10x Genomics, to them and they will demultiplex for you. You will get two fastq files per sample. Read 1 contains the cell barcodes and UMI and Read 2 contains the reads from cDNA.

If you sequence by yourself, you need to run bcl2fastq by yourself with a SampleSheet.csv. Here is an example of SampleSheet.csv of a NextSeq run with two different samples using the indexing primers from the A1 and B1 wells, respectively:

[Header],,,,,,,,,,,
IEMFileVersion,5,,,,,,,,,,
Date,17/12/2019,,,,,,,,,,
Workflow,GenerateFASTQ,,,,,,,,,,
Application,NextSeq FASTQ Only,,,,,,,,,,
Instrument Type,NextSeq/MiniSeq,,,,,,,,,,
Assay,AmpliSeq Library PLUS for Illumina,,,,,,,,,,
Index Adapters,AmpliSeq CD Indexes (384),,,,,,,,,,
Chemistry,Amplicon,,,,,,,,,,
,,,,,,,,,,,
[Reads],,,,,,,,,,,
28,,,,,,,,,,,
98,,,,,,,,,,,
,,,,,,,,,,,
[Settings],,,,,,,,,,,
,,,,,,,,,,,
[Data],,,,,,,,,,,
Sample_ID,Sample_Name,Sample_Plate,Sample_Well,Index_Plate,Index_Plate_Well,I7_Index_ID,index,I5_Index_ID,index2,Sample_Project,Description
Sample01,,,,,,SI-GA-A1_1,GGTTTACT,,,,
Sample01,,,,,,SI-GA-A1_2,CTAAACGG,,,,
Sample01,,,,,,SI-GA-A1_3,TCGGCGTC,,,,
Sample01,,,,,,SI-GA-A1_4,AACCGTAA,,,,
Sample02,,,,,,SI-GA-B1_1,GTAATCTT,,,,
Sample02,,,,,,SI-GA-B1_2,TCCGGAAG,,,,
Sample02,,,,,,SI-GA-B1_3,AGTTCGGC,,,,
Sample02,,,,,,SI-GA-B1_4,CAGCATCA,,,,

You can see each sample actually has four different index sequences. This is because each well from the plate PN-1000213/PN-2000240 actually contain four different indices for base balancing. You can also use dual index (PN-1000215/PN-3000431), and you should add that to the SampleSheet.csv if you use that. Simply run bcl2fastq like this:

bcl2fastq --no-lane-splitting \
          --ignore-missing-positions \
          --ignore-missing-controls \
          --ignore-missing-filter \
          --ignore-missing-bcls \
          -r 4 -w 4 -p 4

After this, you will have R1_001.fastq.gz and R2_001.fastq.gz for each sample:

# sample01
Sample01_S1_R1_001.fastq.gz # 28 bp: cell barcode + UMI
Sample01_S1_R2_001.fastq.gz # cDNA reads

# sample02
Sample02_S2_R1_001.fastq.gz # 28 bp: cell barcode + UMI
Sample02_S2_R2_001.fastq.gz # cDNA reads

You are good to go from here.

Public Data#

For the purpose of demonstration, we will use the 10x Genomics Single Cell 3’ V3 data from the following paper:

Note

Mereu E, Lafzi A, Moutinho C, Ziegenhain C, McCarthy DJ, Álvarez-Varela A, Batlle E, Sagar, Grün D, Lau JK, Boutet SC, Sanada C, Ooi A, Jones RC, Kaihara K, Brampton C, Talaga Y, Sasagawa Y, Tanaka K, Hayashi T, Braeuning C, Fischer C, Sauer S, Trefzer T, Conrad C, Adiconis X, Nguyen LT, Regev A, Levin JZ, Parekh S, Janjic A, Wange LE, Bagnoli JW, Enard W, Gut M, Sandberg R, Nikaido I, Gut I, Stegle O, Heyn H (2020) Benchmarking single-cell RNA-sequencing protocols for cell atlas projects. Nat Biotechnol 38:747–755. https://doi.org/10.1038/s41587-020-0469-4

where the authors benchmarked quite a few different scRNA-seq methods using a standardised sample: a mixture of different human, mouse and dog cells. We are going to use the data from the 10x Genomics Single Cell 3’ V3 method. You can download the fastq file from this ENA page. There are two runs, but I’m just downloading the first run for the demonstration.

mkdir -p mereu2020/10xV3
wget -P mereu2020/10xV3 -c ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR105/009/SRR10587809/SRR10587809_1.fastq.gz
wget -P mereu2020/10xV3 -c ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR105/009/SRR10587809/SRR10587809_2.fastq.gz

Prepare Whitelist#

The barcodes on the gel beads of the 10x Genomics platform are well defined. We need the information for the V3 chemistry. If you have cellranger in your computer, you will find a file called 3M-february-2018.txt.gz in the lib/python/cellranger/barcodes/ directory. If you don’t have cellranger, I have prepared the file for you:

# download the whitelist
wget -P mereu2020/10xV3 wget https://teichlab.github.io/scg_lib_structs/data/10X-Genomics/3M-february-2018.txt.gz
gunzip mereu2020/10xV3/3M-february-2018.txt.gz

From FastQ To Count Matrix#

Now we could start the preprocessing by simply doing:

STAR --runThreadN 4 \
     --genomeDir mix_hg38_mm10/star_index \
     --readFilesCommand zcat \
     --outFileNamePrefix mereu2020/star_outs/ \
     --readFilesIn mereu2020/10xV3/SRR10587809_2.fastq.gz mereu2020/10xV3/SRR10587809_1.fastq.gz \
     --soloType CB_UMI_Simple \
     --soloCBstart 1 --soloCBlen 16 --soloUMIstart 17 --soloUMIlen 12 \
     --soloCBwhitelist mereu2020/10xV3/3M-february-2018.txt \
     --soloCellFilter EmptyDrops_CR \
     --soloStrand Forward \
     --outSAMattributes CB UB \
     --outSAMtype BAM SortedByCoordinate

Explanation#

If you understand the 10x Genomics Single Cell 3’ V3 experimental procedures described in this GitHub Page, the command above should be straightforward to understand.

--runThreadN 4

Use 4 cores for the preprocessing. Change accordingly if using more or less cores.

--genomeDir mix_hg38_mm10/star_index

Pointing to the directory of the star index. The public data from the above paper was produced using the HCA reference sample, which consists of human PBMCs (60%), and HEK293T (6%), mouse colon (30%), NIH3T3 (3%) and dog MDCK cells (1%). Therefore, we need to use the species mixing reference genome. We also need to add the dog genome, but the dog cells only take 1% of all cells, so I did not bother in this documentation.

--readFilesCommand zcat

Since the fastq files are in .gz format, we need the zcat command to extract them on the fly.

--outFileNamePrefix mereu2020/star_outs/

We want to keep everything organised. This directs all output files inside the mereu2020/star_outs directory.

--readFilesIn mereu2020/10xV3/SRR10587809_2.fastq.gz mereu2020/10xV3/SRR10587809_1.fastq.gz

If you check the manual, we should put two files here. The first file is the reads that come from cDNA, and the second the file should contain cell barcode and UMI. In 10x Genomics Single Cell 3’ V3, cDNA reads come from Read 2, and the cell barcode and UMI come from Read 1. Check the 10x Genomics Single Cell 3’ V3 GitHub Page if you are not sure.

--soloType CB_UMI_Simple

Most of the time, you should use this option, and specify the configuration of cell barcodes and UMI in the command line (see immediately below). Sometimes, it is actually easier to prepare the cell barcode and UMI file upfront so that we could use this parameter.

--soloCBstart 1 --soloCBlen 16 --soloUMIstart 17 --soloUMIlen 12

The name of the parameter is pretty much self-explanatory. If using --soloType CB_UMI_Simple, we can specify where the cell barcode and UMI start and how long they are in the reads from the first file passed to --readFilesIn. Note the position is 1-based (the first base of the read is 1, NOT 0).

--soloCBwhitelist mereu2020/10xV3/3M-february-2018.txt

The plain text file containing all possible valid cell barcodes, one per line. 10x Genomics Single Cell 3’ V3 is a commercial platform. The whitelist is taken from their commercial software cellranger.

--soloCellFilter EmptyDrops_CR

Experiments are never perfect. Even for droplets that do not contain any cell, you may still get some reads. In general, the number of reads from those droplets should be much smaller, often orders of magnitude smaller, than those droplets with cells. In order to identify true cells from the background, you can apply different algorithms. Check the star manual for more information. We use EmptyDrops_CR which is the most frequently used parameter.

--soloStrand Forward

The choice of this parameter depends on where the cDNA reads come from, i.e. the reads from the first file passed to --readFilesIn. You need to check the experimental protocol. If the cDNA reads are from the same strand as the mRNA (the coding strand), this parameter will be Forward (this is the default). If they are from the opposite strand as the mRNA, which is often called the first strand, this parameter will be Reverse. In the case of 10x Genomics Single Cell 3’ V3, the cDNA reads are from the Read 2 file. During the experiment, the mRNA molecules are captured by barcoded oligo-dT primer containing UMI and the Illumina Read 1 sequence. Therefore, Read 1 consists of cell barcodes and UMI comes from the first strand, complementary to the coding strand. Read 2 comes from the coding strand. Therefore, use Forward for 10x Genomics Single Cell 3’ V3 data. This Forward parameter is the default, because many protocols generate data like this, but I still specified it here to make it clear. Check the 10x Genomics Single Cell 3’ V3 GitHub Page if you are not sure.

--outSAMattributes CB UB

We want the cell barcode and UMI sequences in the CB and UB attributes of the output, respectively. The information will be very helpful for downstream analysis.

--outSAMtype BAM SortedByCoordinate

We want sorted BAM for easy handling by other programs.

If everything goes well, your directory should look the same as the following:

scg_prep_test/mereu2020/
├── 10xV3
│   ├── 3M-february-2018.txt
│   ├── SRR10587809_1.fastq.gz
│   └── SRR10587809_2.fastq.gz
└── star_outs
    ├── Aligned.sortedByCoord.out.bam
    ├── Log.final.out
    ├── Log.out
    ├── Log.progress.out
    ├── SJ.out.tab
    └── Solo.out
        ├── Barcodes.stats
        └── Gene
            ├── Features.stats
            ├── filtered
            │   ├── barcodes.tsv
            │   ├── features.tsv
            │   └── matrix.mtx
            ├── raw
            │   ├── barcodes.tsv
            │   ├── features.tsv
            │   └── matrix.mtx
            ├── Summary.csv
            └── UMIperCellSorted.txt

6 directories, 18 files

10x Genomics Single Cell 3’ V3

Contents