ASAP-seq; Multiplexed CRISPR Perturbations in Primary T Cells

Dataset: ASAP-seq: Multiplexed CRISPR Perturbations in Primary T Cells

Mimitou, E.P., Lareau, C.A., Chen, K.Y., Zorzetto-Fernandes, A.L., Hao, Y., Takeshima, Y., Luo, W., Huang, T.-S., Yeung, B.Z., Papalexi, E., et al. (2021). Scalable, multimodal profiling of chromatin accessibility, gene expression and protein levels in single cells. Nat. Biotechnol. 39, 1246–1258.


Preparation

Download fastq files from Gene Expression Omnibus.

$ ls -l

SRR12476627_1.fastq.gz
SRR12476627_2.fastq.gz
SRR12476627_3.fastq.gz
SRR12476628_1.fastq.gz
SRR12476628_2.fastq.gz
SRR12476628_3.fastq.gz
SRR12476630_1.fastq.gz
SRR12476630_2.fastq.gz
SRR12476630_3.fastq.gz
SRR12476631_1.fastq.gz
SRR12476631_2.fastq.gz
SRR12476631_3.fastq.gz

Hashtag oligos

$ cat SRR12476630_2.fastq.gz SRR12476631_2.fastq.gz > hto_read_2.fq.gz

$ cat SRR12476630_3.fastq.gz SRR12476631_3.fastq.gz > hto_read_3.fq.gz

Antibody-derived tags

$ cat SRR12476627_2.fastq.gz SRR12476628_2.fastq.gz > adt_read_2.fq.gz

$ cat SRR12476627_3.fastq.gz SRR12476628_3.fastq.gz > adt_read_3.fq.gz

Download cell barcode info from the manuscript’s wonderful GitHub repository. We need to get the cell-associated barcodes of this single-cell ATAC-Seq library.

$ wget https://github.com/caleblareau/asap_reproducibility/blob/master/CD4_CRISPR_asapseq/data/filtered_peak_bc_matrix.h5

$ ls -l

filtered_peak_bc_matrix.h5

Retrieve cell-associated barcodes from this downloaded h5 file generated by 10x Genomics’ Cell Ranger ATAC.

R version 4.1.3 (2022-03-10) -- "One Push-Up"
Platform: aarch64-apple-darwin21.3.0 (64-bit)

r$> h5f <- rhdf5::H5Fopen(name = "filtered_peak_bc_matrix.h5")

r$> cell_barcodes <- h5f$matrix$barcodes

r$> rhdf5::H5Fclose(h5f)

r$> write.table(
        x = cell_barcodes,
        file = "cell_barcodes.txt",
        col.names = FALSE,
        row.names = FALSE,
        quote = FALSE
    )

Inspect cell barcodes.

$ head cell_barcodes.txt

AAACGAAAGCTCGTTA-1
AAACGAAAGCTGAGGT-1
AAACGAAAGGTGAACC-1
AAACGAACAACATAAG-1
AAACGAACAATAGCGG-1
AAACGAACAATCCATG-1
AAACGAACACGTTAGT-1
AAACGAACAGAGATGC-1
AAACGAACAGCGTAGA-1
AAACGAAGTAAACGAT-1

Prepare feature barcodes (hashtag oligos, HTOs) from sheet ‘Supplementary_1_Perturbation’ in the supplementary table file.

$ cat feature_barcodes_HTO.tsv

anti-human_hashtag_1    GTCAACTCTTTAGCG
anti-human_hashtag_2    TGATGGCCTATTGGG
anti-human_hashtag_3    TTCCGCCTCTCTTTG
anti-human_hashtag_4    AGTAAGTTCAGCGTA
anti-human_hashtag_5    AAGTATCGTTTCGCA
anti-human_hashtag_12   TAACGACCAGCCATA
anti-human_hashtag_13   AAATCTCTCAGGCTC

Prepare feature barcodes (antibody-derived tags, ADTs) from sheet ‘Supplementary_1_Hashing’ in the supplementary table file.

$ cat feature_barcodes_ADT.tsv

UCHT1   CTCATTGTAACTCCT
RPA-T4  TGTTCCCGCTCAACT
SK1     GCGCAACTTGATGAT
HI100   TCAATCCTTCCGCTT
UCHL1   CTCCGAATCATGTTG
EH12.2H7        ACAGCGCCGTATTTA
A019D5  GTGTGTTGTCCTATG
DX2     CCAGCTCATTAGAGC
G043H7  AGTTCAGTCAACCGA
WM59    ACCTTTATGCCACGG
BC96    TTTGTCCTGTACGCC
QA17A04 AACTCCCTATGGAGG
FN50    GTCTCTTGGCTTAAA
O323    GCACTCCTGCATGTA
C398.4A CGCGCACCCATTAAA
BJ18    AATCCTTCCGAATGT
A15153G TTGCTTACCGCCAGA
CD7-6B7 TGGATTCCCGGACTT
Ber-ACT35_(ACT35)       AACCCACCGTTGTTA
DREG-56 GTCCCTGCAACTTGA
A1      TTACCTGGTATCCGT
4B4-1   CAGTAAGTTCGGGAC
108-17  ACCTTTCGACACTCG
CD28.2  TGAGAACGACCCTAA
HIT2    TGTACCCGCTTGTGA
AD2     CAGTTCCTCAGTTCG
HP-3G10 GTACGCAGTCCTTCT
HIB19   CTGGGCAATTACTCG
2D1     TCCCTTGCGATTTAC
M5E2    TCTCAGACCTCCGTA
5.1H11  TCCTTTCCTGATAGG
3G8     AAGTTCACTCTTTGC
IP26    CGTAACGTAGAGCGA
F38-2E2 TGTCCTACCCAACTT
J252D4  AATTCAACCGTCGCC
S-HCL-3 TACGCCTATAACTTG
MOPC-21 GCCGGACGACATTAA

Cell hashing

QC

The first 100,000 read pairs are sampled (default, set by -n) for quality control. By default, diagnostic results and plots are generated in the qc directory (set by --output_directory), and the full length of read 1 and read 2 are searched against reference cell and feature barcodes, respectively. The per base content of both read pairs and the distribution of matched barcode positions are summarized. Use -r1_c and/or -r2_c to limit the search range, and -cb_n and/or -fb_n to set the mismatch tolerance for cell and/or feature barcode matching (default 3).

This library was constructed using 10x Genomics’ Chromium Single Cell ATAC Reagent Kits, where the 16-base pair cell barcode is sequenced during the i5 index read. In Cell Ranger ATAC, the raw 16 bp sequences may be transformed into their reverse-complement counterparts as cell barcodes in the outputs. To achieve the same result in fba, use -cb_rc to reverse-complement the cell barcode sequences.

$ fba qc \
    -1 hto_read_2.fq.gz \
    -2 hto_read_3.fq.gz \
    -w cell_barcodes.txt \
    -f feature_barcodes_HTO.tsv \
    --output_directory qc \
    -cb_rc

This library was constructed using the Chromium Single Cell ATAC Reagent Kits and sequenced on the Illumina NextSeq 550 platform. The base composition analysis reveals that the cell barcodes in read 2 are enriched for A bases.

../../../_images/Pyplot_read1_per_base_seq_content_HTO.webp

The per base content of read 3 suggests that the feature barcodes are located in bases 0-14, as indicated by the distribution of matched barcode positions (See the fba qc results for more details).

../../../_images/Pyplot_read2_per_base_seq_content_HTO.webp ../../../_images/Pyplot_read2_barcodes_starting_ending_HTO.webp

The detailed qc results are stored in the feature_barcoding_output.tsv.gz file. The matching_pos columns indicate the matched positions on reads, while the matching_description columns indicate mismatches in the format of substitutions:insertions:deletions.

$ gzip -dc feature_barcoding_output.tsv.gz | grep -v no | head

read1_seq       cell_barcode    cb_matching_pos cb_matching_description read2_seq       feature_barcode fb_matching_pos fb_matching_description
TTTAAGCTGCCTAACA        TTGTTAGCTGCCCAAC        0:15    2:0:1   TAACGACCAGCCATNNNANAANNANNANNNANNNNANNNNNANNNNNNNANNNNNNNNNNNNNNNNNANNNN        anti-human_hashtag_12_TAACGACCAGCCATA   0:15       1:0:0
AGAACGCGAAAAGGTT        AGAACGCGAGTACGTT        0:16    3:0:0   TGATGACCTATTGGNNNAAAANNANNANNNANNNNANANNNANNNANNNANNNNNNANNNNNNNNNNANNAN        anti-human_hashtag_2_TGATGGCCTATTGGG    0:15       2:0:0
TGAGACTTGGCAGGAT        TGAGACTTGGCAGGAT        0:16    0:0:0   TTTCGCCTTTCTTTGNAAAAANNANNAANAANNANANANAAANNAAANNAANANNAAAAANNANNNNAANAN        anti-human_hashtag_3_TTCCGCCTCTCTTTG    0:15       2:0:0
ATTTATTGACGCAAAG        CTTATTGTGCGCAAAG        1:16    2:0:1   TTCCACCTCTCTTTGNAAAAAANAANAAAAANAANAAANAAANAAAANAAAAANNAAAAAANAANNNAAAAN        anti-human_hashtag_3_TTCCGCCTCTCTTTG    0:15       1:0:0
CGCCCTTCTGGGTAGT        CGCCCTTCTGGGTAGT        0:16    0:0:0   AAGTATCGTTTCGCATAAAAAAAAANAAAAANAANAAANAAAAAAAAAAAAAAAAAAAAAANAAANNAAAAN        anti-human_hashtag_5_AAGTATCGTTTCGCA    0:15       0:0:0
TACCTCGACCTGGAAG        TACCTCGACCTGGAAG        0:16    0:0:0   ATCAACTCTTTAGCGCAAAAAAAAAAAAAAAAAAAAAANAAAAAAAAAAAAAAAAAAAAAANAAAANAAAAN        anti-human_hashtag_1_GTCAACTCTTTAGCG    0:15       1:0:0
ACCACCCCCAACACCC        TACCACCACCCTAACA        0:13    0:0:3   ATCAACTCTTTAACATAAAAAAAAAAAAAAAAAAAAAANAAAAAAAAAAAAAAAAAAAAAANAAAAAAAAAA        anti-human_hashtag_1_GTCAACTCTTTAGCG    0:15       3:0:0
GCTAACTGATTCGGGC        AACTCGATGTCGGGCT        3:16    0:0:3   TAACAACCAGCCATAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACAAAAAAAAAAAANAAAAAAAAAA        anti-human_hashtag_12_TAACGACCAGCCATA   0:15       1:0:0
TTTGCGGCTCTCCTAC        TTTGCGGCTCATGCAT        0:14    1:0:2   TTCCACCTCTCTTTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAANAAAAAAAAAA        anti-human_hashtag_3_TTCCGCCTCTCTTTG    0:15       1:0:0

Barcode extraction

The lengths of cell and feature barcodes are all identical (16 and 15, respectively). And based on the qc results, the distributions of starting and ending positions of cell and feature barcodes are very uniform. Search ranges are set to 0,16 on read 2 and 0,15 on read 3. One mismatch for cell and feature barcodes (-cb_m, -cf_m) are allowed. And by default, three ambiguous nucleotides (Ns) for read 1 and read 2 (-cb_n, -cf_n) are allowed. Use -cb_rc to reverse-complement cell barcode sequences.

$ fba extract \
    -1 hto_read_2.fq.gz \
    -2 hto_read_3.fq.gz \
    -w cell_barcodes.txt \
    -f feature_barcodes_HTO.tsv \
    -o feature_barcoding_output_HTO.tsv.gz \
    -r1_c 0,16 \
    -r2_c 0,15 \
    -cb_m 1 \
    -fb_m 1 \
    -cb_n 3 \
    -fb_n 3 \
    -cb_rc

Preview of result.

$ gzip -dc feature_barcoding_output_HTO.tsv.gz | head

read1_seq       cell_barcode    cb_num_mismatches       read2_seq       feature_barcode fb_num_mismatches
AATAACCGACAGGTGA        AATCACCGACAGGTGA        1       ATCAACTCTTTAGCGtaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaanaaaaaaaaaa        anti-human_hashtag_1_GTCAACTCTTTAGCG    1
TGCAGTATGCCTCGTA        TGCAGTATGCCTCGTT        1       TAATGGCCTATTGGGgaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaacacccaaaaaaaaaaaaaaa        anti-human_hashtag_2_TGATGGCCTATTGGG    1
TCGCGGTGAGCTTACA        TCGCGGTGAGCTTACA        0       TTCCGCCTCTCTTTGcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa        anti-human_hashtag_3_TTCCGCCTCTCTTTG    0
AACTAGCACTATTGCG        AACTAGCACTATTGCG        0       AAGTATCGTTTCGCAcaaaaaaaaaaaaaaaaaaaaaaaaataacacttaaaaataaaaaaaaaaaacccaa        anti-human_hashtag_5_AAGTATCGTTTCGCA    0
TGCAATGTGGGGTTCC        TGCAATGTGGGGTTCC        0       TTCCGCCTCTCTTTGaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa        anti-human_hashtag_3_TTCCGCCTCTCTTTG    0
TGGATAGCTATCTGTG        TGGATAGCTATCTGTG        0       AAGTATCGTTTCGCAcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaacccaaaaaaaaaaaaaaa        anti-human_hashtag_5_AAGTATCGTTTCGCA    0
AGCAGAGACATCCTAG        AGCAGAGACATCCTAG        0       TTCCGCCTCTCTTTGaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa        anti-human_hashtag_3_TTCCGCCTCTCTTTG    0
CTTAATCTGTGTTGTG        CTTAATCTGTGTTGTG        0       AAGTATCGTTTCGCAcaaaaaaaaaaaaggtgtattactgtctcttatacacatctgacgctgccgacgact        anti-human_hashtag_5_AAGTATCGTTTCGCA    0
GTTTCATTGTGGCATT        GTTTCATTGTGGCATT        0       AATAAGTTCAGCGTAgaaaaaaaaaaaaaaaaaaaaaaaaaatttaaatttgaattaaaataaaaaaaaata        anti-human_hashtag_4_AGTAAGTTCAGCGTA    1

Result summary.

72.4% (22,820,698 out of 31,512,084) of total read pairs have valid cell and feature barcodes.

2022-03-16 00:14:08,601 - fba.__main__ - INFO - fba version: 0.0.x
2022-03-16 00:14:08,601 - fba.__main__ - INFO - Initiating logging ...
2022-03-16 00:14:08,601 - fba.__main__ - INFO - Python version: 3.10
2022-03-16 00:14:08,601 - fba.__main__ - INFO - Using extract subcommand ...
2022-03-16 00:14:08,635 - fba.levenshtein - INFO - Number of reference cell barcodes: 9,151
2022-03-16 00:14:08,635 - fba.levenshtein - INFO - Number of reference feature barcodes: 7
2022-03-16 00:14:08,635 - fba.levenshtein - INFO - Read 1 coordinates to search: [0, 16)
2022-03-16 00:14:08,635 - fba.levenshtein - INFO - Read 2 coordinates to search: [0, 15)
2022-03-16 00:14:08,635 - fba.levenshtein - INFO - Cell barcode maximum number of mismatches: 1
2022-03-16 00:14:08,635 - fba.levenshtein - INFO - Feature barcode maximum number of mismatches: 1
2022-03-16 00:14:08,635 - fba.levenshtein - INFO - Read 1 maximum number of N allowed: 3
2022-03-16 00:14:08,635 - fba.levenshtein - INFO - Read 2 maximum number of N allowed: 3
2022-03-16 00:14:08,871 - fba.levenshtein - INFO - Matching ...
2022-03-16 00:19:01,333 - fba.levenshtein - INFO - Read pairs processed: 10,000,000
2022-03-16 00:23:44,891 - fba.levenshtein - INFO - Read pairs processed: 20,000,000
2022-03-16 00:28:29,304 - fba.levenshtein - INFO - Read pairs processed: 30,000,000
2022-03-16 00:29:12,889 - fba.levenshtein - INFO - Number of read pairs processed: 31,512,084
2022-03-16 00:29:12,890 - fba.levenshtein - INFO - Number of read pairs w/ valid barcodes: 22,820,698
2022-03-16 00:29:12,902 - fba.__main__ - INFO - Done.

Matrix generation

Only fragments with correctly matched cell and feature barcodes are included. Use -ul to set the UMI length (default 12). Setting to 0 means no UMIs and read counts are summarized instead. Use -cb_rc to reverse-complement cell barcode sequences in the output matrix if needed.

The generated feature count matrix can be easily imported into well-established single cell analysis packages such as Seurat and Scanpy.

$ fba count \
    -i feature_barcoding_output_HTO.tsv.gz \
    -o matrix_featurecount_HTO.csv.gz \
    -ul 0

Result summary.

The median number of reads per cell of this HTO library is 1,893.0.

2022-03-16 00:29:13,026 - fba.__main__ - INFO - fba version: 0.0.x
2022-03-16 00:29:13,026 - fba.__main__ - INFO - Initiating logging ...
2022-03-16 00:29:13,026 - fba.__main__ - INFO - Python version: 3.10
2022-03-16 00:29:13,026 - fba.__main__ - INFO - Using count subcommand ...
2022-03-16 00:29:14,943 - fba.count - INFO - UMI-tools version: 1.1.2
2022-03-16 00:29:14,950 - fba.count - INFO - UMI length set to 0, ignoring UMI information. Skipping arguments: "-us/--umi_start".
2022-03-16 00:29:14,950 - fba.count - INFO - Header: read1_seq cell_barcode cb_num_mismatches read2_seq feature_barcode fb_num_mismatches
2022-03-16 00:29:52,721 - fba.count - INFO - Number of read pairs processed: 22,820,698
2022-03-16 00:29:52,730 - fba.count - INFO - Number of cell barcodes detected: 9,151
2022-03-16 00:29:52,730 - fba.count - INFO - Number of features detected: 7
2022-03-16 00:29:52,730 - fba.count - INFO - Counting ...
2022-03-16 00:29:52,940 - fba.count - INFO - Total reads: 22,820,698
2022-03-16 00:29:52,941 - fba.count - INFO - Median number of reads per cell: 1,893.0
2022-03-16 00:29:53,099 - fba.__main__ - INFO - Done.

In [1]: import pandas as pd

In [2]: m = pd.read_csv("matrix_featurecount.csv.gz", index_col=0)

In [3]: m.sum(axis=1)
Out[3]:
anti-human_hashtag_12_TAACGACCAGCCATA    4402031
anti-human_hashtag_13_AAATCTCTCAGGCTC    2225016
anti-human_hashtag_1_GTCAACTCTTTAGCG     4107376
anti-human_hashtag_2_TGATGGCCTATTGGG     2672503
anti-human_hashtag_3_TTCCGCCTCTCTTTG     2469687
anti-human_hashtag_4_AGTAAGTTCAGCGTA     3172034
anti-human_hashtag_5_AAGTATCGTTTCGCA     3772051
dtype: int64

In [4]: m1 = m.loc[
...:     [
...:         "anti-human_hashtag_1_GTCAACTCTTTAGCG",
...:         "anti-human_hashtag_2_TGATGGCCTATTGGG",
...:         "anti-human_hashtag_3_TTCCGCCTCTCTTTG",
...:         "anti-human_hashtag_4_AGTAAGTTCAGCGTA",
...:         "anti-human_hashtag_5_AAGTATCGTTTCGCA",
...:     ],
...:     :,
...: ]

In [5]: m1.to_csv(path_or_buf="matrix_featurecount_HTO_1-5.csv.gz",
                  compression="infer")

In [6]: m2 = m.loc[[
...:     "anti-human_hashtag_12_TAACGACCAGCCATA",
...:     "anti-human_hashtag_13_AAATCTCTCAGGCTC"
...: ], :]

In [7]: m2.to_csv(path_or_buf="matrix_featurecount_HTO_12-13.csv.gz",
                  compression="infer")

Demultiplexing

Gaussian mixture model

The implementation of demultiplexing method 2 (set by -dm) is inspired by the method described on the 10x Genomics’ website. To set the probability threshold for demultiplexing, use -p (default 0.9). To generate visualization plots, set -v.

$ fba demultiplex \
    -i matrix_featurecount_HTO_1-5.csv.gz \
    -dm 2 \
    -v

2022-03-16 00:38:18,749 - fba.__main__ - INFO - fba version: 0.0.x
2022-03-16 00:38:18,749 - fba.__main__ - INFO - Initiating logging ...
2022-03-16 00:38:18,749 - fba.__main__ - INFO - Python version: 3.9
2022-03-16 00:38:18,749 - fba.__main__ - INFO - Using demultiplex subcommand ...
2022-03-16 00:38:21,709 - fba.__main__ - INFO - Skipping arguments: "-q/--quantile", "-cm/--clustering_method"
2022-03-16 00:38:21,709 - fba.demultiplex - INFO - Output directory: demultiplexed
2022-03-16 00:38:21,709 - fba.demultiplex - INFO - Demultiplexing method: 2
2022-03-16 00:38:21,709 - fba.demultiplex - INFO - UMI normalization method: clr
2022-03-16 00:38:21,709 - fba.demultiplex - INFO - Visualization: On
2022-03-16 00:38:21,709 - fba.demultiplex - INFO - Visualization method: tsne
2022-03-16 00:38:21,709 - fba.demultiplex - INFO - Loading feature count matrix: matrix_featurecount_HTO_1-5.csv.gz ...
2022-03-16 00:38:21,796 - fba.demultiplex - INFO - Number of cells: 9,151
2022-03-16 00:38:21,796 - fba.demultiplex - INFO - Number of positive cells for a feature to be included: 200
2022-03-16 00:38:21,810 - fba.demultiplex - INFO - Number of features: 5 / 5 (after filtering / original in the matrix)
2022-03-16 00:38:21,810 - fba.demultiplex - INFO - Features: anti-human_hashtag_1 anti-human_hashtag_2 anti-human_hashtag_3 anti-human_hashtag_4 anti-human_hashtag_5
2022-03-16 00:38:21,810 - fba.demultiplex - INFO - Total UMIs/reads: 16,193,651 / 16,193,651
2022-03-16 00:38:21,817 - fba.demultiplex - INFO - Median number of UMIs/reads per cell: 1,326.0 / 1,326.0
2022-03-16 00:38:21,817 - fba.demultiplex - INFO - Demultiplexing ...
2022-03-16 00:38:24,130 - fba.demultiplex - INFO - Generating heatmap ...
2022-03-16 00:38:26,376 - fba.demultiplex - INFO - Embedding ...
2022-03-16 00:38:43,503 - fba.__main__ - INFO - Done.

Heatmap of the relative abundance of features (hashtag oligos, HTOs) across all cells. Each column represents a single cell. This is a re-creation of Extended Data Fig. 6b in Mimitou, E.P., et al. (2021).

Heatmap

Preview the demultiplexing result: the numbers of singlets, multiplets and negatives are 7,728 (84.4%), 1,224 (13.4%), and 199 (2.2%), respectively.

In [1]: import pandas as pd

In [2]: m = pd.read_csv("demultiplexed/matrix_cell_identity.csv.gz", index_col=0)

In [3]: m.loc[:, m.sum(axis=0) == 1].sum(axis=1)
Out[3]:
anti-human_hashtag_1    1493
anti-human_hashtag_2    1511
anti-human_hashtag_3    1395
anti-human_hashtag_4    1675
anti-human_hashtag_5    1654
dtype: int64

In [4]: sum(m.sum(axis=0) == 1)
Out[4]: 7728

In [5]: sum(m.sum(axis=0) > 1)
Out[5]: 1224

In [6]: sum(m.sum(axis=0) == 0)
Out[6]: 199

In [7]: m.shape
Out[7]: (5, 9151)

t-SNE embedding of cells based on the abundance of features (phage-derived tags, no transcriptome information used). Colors indicate the hashtag status for each cell, as called by FBA.

t-SNE embedding 

$ fba demultiplex \
    -i matrix_featurecount_HTO_12-13.csv.gz \
    -dm 2 \
    -v

2022-03-16 00:39:44,380 - fba.__main__ - INFO - Initiating logging ...
2022-03-16 00:39:44,380 - fba.__main__ - INFO - Python version: 3.9
2022-03-16 00:39:44,380 - fba.__main__ - INFO - Using demultiplex subcommand ...
2022-03-16 00:39:47,238 - fba.__main__ - INFO - Skipping arguments: "-q/--quantile", "-cm/--clustering_method"
2022-03-16 00:39:47,238 - fba.demultiplex - INFO - Output directory: demultiplexed
2022-03-16 00:39:47,238 - fba.demultiplex - INFO - Demultiplexing method: 2
2022-03-16 00:39:47,238 - fba.demultiplex - INFO - UMI normalization method: clr
2022-03-16 00:39:47,238 - fba.demultiplex - INFO - Visualization: On
2022-03-16 00:39:47,238 - fba.demultiplex - INFO - Visualization method: tsne
2022-03-16 00:39:47,238 - fba.demultiplex - INFO - Loading feature count matrix: matrix_featurecount_HTO_12-13.csv.gz ...
2022-03-16 00:39:47,329 - fba.demultiplex - INFO - Number of cells: 9,151
2022-03-16 00:39:47,341 - fba.demultiplex - INFO - Number of positive cells for a feature to be included: 200
2022-03-16 00:39:47,355 - fba.demultiplex - INFO - Number of features: 2 / 2 (after filtering / original in the matrix)
2022-03-16 00:39:47,363 - fba.demultiplex - INFO - Features: anti-human_hashtag_12 anti-human_hashtag_13
2022-03-16 00:39:47,363 - fba.demultiplex - INFO - Total UMIs/reads: 6,627,047 / 6,627,047
2022-03-16 00:39:47,370 - fba.demultiplex - INFO - Median number of UMIs/reads per cell: 559.0 / 559.0
2022-03-16 00:39:47,370 - fba.demultiplex - INFO - Demultiplexing ...
2022-03-16 00:39:48,484 - fba.demultiplex - INFO - Generating heatmap ...
2022-03-16 00:39:49,412 - fba.demultiplex - INFO - Embedding ...
2022-03-16 00:40:06,551 - fba.__main__ - INFO - Done.

Heatmap of the relative abundance of features (hashtag oligos, HTOs) across all cells. Each column represents a single cell. This is a re-creation of Extended Data Fig. 6b in Mimitou, E.P., et al. (2021).

Heatmap

Preview the demultiplexing result: the numbers of singlets, multiplets and negatives are 7,924 (86.6%), 856 (9.4%), and 371 (4.1%), respectively.

In [1]: import pandas as pd

In [2]: m = pd.read_csv("demultiplexed/matrix_cell_identity.csv.gz", index_col=0)

In [3]: m.loc[:, m.sum(axis=0) == 1].sum(axis=1)
Out[3]:
anti-human_hashtag_12    4018
anti-human_hashtag_13    3906
dtype: int64

In [4]: [sum(m.sum(axis=0) == i) for i in (1, 2, 0)]
Out[4]: [7924, 856, 371]

In [5]: m.shape
Out[5]: (2, 9151)

t-SNE embedding of cells based on the abundance of features (phage-derived tags, no transcriptome information used). Colors indicate the hashtag status for each cell, as called by FBA.

t-SNE embedding

CITE-seq

QC

Same as the HTO library, sample the first 10,000 (set by -n) read pairs for quality control.

$ fba qc \
    -1 adt_read_2.fq.gz \
    -2 adt_read_3.fq.gz \
    -w cell_barcodes.txt \
    -f feature_barcodes_HTO.tsv \
    --output_directory qc \
    -cb_rc

Cell barcodes are A-rich.

../../../_images/Pyplot_read1_per_base_seq_content_ADT.webp

As for read 3, based on the per base content, it suggests that bases 0-14 are actually our feature barcodes (See the distribution of matched barcode positions on read 3).

../../../_images/Pyplot_read2_per_base_seq_content_ADT.webp ../../../_images/Pyplot_read2_barcodes_starting_ending_ADT.webp

The detailed qc results are stored in feature_barcoding_output.tsv.gz file. matching_pos columns indicate the matched positions on reads. matching_description columns indicate mismatches in substitutions:insertions:deletions format.

$ gzip -dc feature_barcoding_output.tsv.gz | grep -v no | head

read1_seq       cell_barcode    cb_matching_pos cb_matching_description read2_seq       feature_barcode fb_matching_pos fb_matching_description
CCTAAGAAAAAGCTGC        CCTAAGAACAAGACAT        0:14    1:0:2   AATCCTTCCGAATNNNNNNANNNNNNANNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN        BJ18_AATCCTTCCGAATGT    0:15    2:0:0
TTATCCAAGGACTGTT        TCCCACATGGACTGTT        3:16    0:0:3   TATTCCCGCTCAANNNNNNANNNNNNANNNANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN        RPA-T4_TGTTCCCGCTCAACT  0:15    3:0:0
GAGAAGAGACCGATTA        AGGAAGAGACCCGAGT        1:15    1:0:2   CTCATTGTAACTCCNNNANAANNANNANNNANNNNANNNNNANNNNNNNANNNNNNNNNNNNNNNNNANNNN        UCHT1_CTCATTGTAACTCCT   0:15    1:0:0
ATACATCATAAACAAA        ATACATCCTTAACGAA        0:16    3:0:0   ATCCTTCCGAATGTNNNAAAANNANNANNAANNNNANANNNANNNANNNANNNNNNANNNNNANNNNANNAN        BJ18_AATCCTTCCGAATGT    0:14    0:0:1
GATCATGTGCTTTGTT        TGAACTGTGCTTTGTT        0:16    1:1:1   CACACACCCATTAAANAAAAANNANNANNAANNNNANANNNANNNANNNAANNNNNAAAANNANNNNAANAN        C398.4A_CGCGCACCCATTAAA 0:15    2:0:0
AACACTCGACAGGTGA        AACACTCGAGAAGGCT        0:14    1:0:2   ATCCCTGCAACTTGANAAAAANNANNAANAANNANANANNNANNNANNNAANANNNAAAANNANNNNAANAN        DREG-56_GTCCCTGCAACTTGA 0:15    1:0:0
ACTGTTCGACTACTCC        AGTGTTCGACTATCTT        0:15    2:0:1   TCCCTTGCGATTTACNAAAAAANAANAANAANAANAAANAAANCAAANNAANANNAAAAANNANNNNAANAN        2D1_TCCCTTGCGATTTAC     0:15    0:0:0
ACTTATCTGTGCATCA        ACTTATCTGTGCATCA        0:16    0:0:0   ACCTTTATGCCACGGNAAAAAANAANAAAAANAANAAANAAANAAAANTTTAANNAAAAAANAANNNAAAAN        WM59_ACCTTTATGCCACGG    0:15    0:0:0
GAGCATAATTTAGCAG        GAGCATACTTGAGCAG        0:16    2:0:0   CAATAAGTTCGGGACCAAAAAAAAANAAAAANAANAAANAAANAAAAAAAAAANAAAAAAANAAANNAAAAN        4B4-1_CAGTAAGTTCGGGAC   0:15    1:0:0

Barcode extraction

The lengths of cell and feature barcodes are all identical (16 and 15, respectively). And based on the qc results, the distributions of starting and ending positions of cell and feature barcodes are very uniform. Search ranges are set to 0,16 on read 2 and 0,15 on read 3. One mismatch for cell and feature barcodes (-cb_m, -cf_m) are allowed. And by default, three ambiguous nucleotides (Ns) for read 1 and read 2 (-cb_n, -cf_n) are allowed. Use -cb_rc to reverse-complement cell barcode sequences for processing.

$ fba extract \
    -1 adt_read_2.fq.gz \
    -2 adt_read_3.fq.gz \
    -w cell_barcodes.txt \
    -f feature_barcodes_ADT.tsv \
    -o feature_barcoding_output_ADT.tsv.gz \
    -r1_c 0,16 \
    -r2_c 0,15 \
    -cb_m 1 \
    -fb_m 1 \
    -cb_n 3 \
    -fb_n 3 \
    -cb_rc

Preview of result.

$ gzip -dc feature_barcoding_output_ADT.tsv.gz | head

read1_seq       cell_barcode    cb_num_mismatches       read2_seq       feature_barcode fb_num_mismatches
CAAGAAATGCATTCAG        CAAGAAATGCATTCAG        0       TATACCCGCTTGTGAtaaaaaaaaaaaaaaaaaaaaaanaaaaaaaaaaaaaaaaaaaaaanaaaaaaaaaa        HIT2_TGTACCCGCTTGTGA    1
GTGGCTGTGTTTGTCT        GTGGCTGTGTTTGTCT        0       TAGATTCCCGGACTTgaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaanaaaaaaaaaa        CD7-6B7_TGGATTCCCGGACTT 1
ACTGATTCTGCCCTAG        ACTGATTCTGCCCTAG        0       TAGATTCCCGGACTTtaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaanaaaaaaaaaa        CD7-6B7_TGGATTCCCGGACTT 1
CCTGCTATGCTGCGGT        CCTGCTATGCTGCGGT        0       TAGATTCCCGGACTTgaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaanaaaaaaaaaa        CD7-6B7_TGGATTCCCGGACTT 1
TGTAGTTTGGATAGCG        GGTAGTTTGGATAGCG        1       TTTATCCTGTACGCCtaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaanaaaaaaaaaa        BC96_TTTGTCCTGTACGCC    1
TCCTTAAACCATCCTC        TCCTTAAACCATCCTC        0       AATCCTTCCGAATGTtaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaanaaaaaaaaaa        BJ18_AATCCTTCCGAATGT    0
TTCCCATCTGGAATAT        TTCCCATCTGGAATAT        0       CTCATTGTAACTCCTcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa        UCHT1_CTCATTGTAACTCCT   0
ACTCAGAACGAATTGG        ACTCAGAACGAATTGA        1       TATTCCCGCTCAACTcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa        RPA-T4_TGTTCCCGCTCAACT  1
CAGGACCTGCGCACTG        CAGGACCTGCGCACTG        0       TATTCCCGCTCAACTcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa        RPA-T4_TGTTCCCGCTCAACT  1

Result summary.

51.3% (27,719,537 out of 54,024,324) of total read pairs have valid cell and feature barcodes.

2022-03-15 23:43:13,501 - fba.__main__ - INFO - fba version: 0.0.x
2022-03-15 23:43:13,501 - fba.__main__ - INFO - Initiating logging ...
2022-03-15 23:43:13,501 - fba.__main__ - INFO - Python version: 3.10
2022-03-15 23:43:13,501 - fba.__main__ - INFO - Using extract subcommand ...
2022-03-15 23:43:13,562 - fba.levenshtein - INFO - Number of reference cell barcodes: 9,151
2022-03-15 23:43:13,562 - fba.levenshtein - INFO - Number of reference feature barcodes: 37
2022-03-15 23:43:13,562 - fba.levenshtein - INFO - Read 1 coordinates to search: [0, 16)
2022-03-15 23:43:13,562 - fba.levenshtein - INFO - Read 2 coordinates to search: [0, 15)
2022-03-15 23:43:13,562 - fba.levenshtein - INFO - Cell barcode maximum number of mismatches: 1
2022-03-15 23:43:13,562 - fba.levenshtein - INFO - Feature barcode maximum number of mismatches: 1
2022-03-15 23:43:13,562 - fba.levenshtein - INFO - Read 1 maximum number of N allowed: 3
2022-03-15 23:43:13,562 - fba.levenshtein - INFO - Read 2 maximum number of N allowed: 3
2022-03-15 23:43:13,798 - fba.levenshtein - INFO - Matching ...
2022-03-15 23:47:34,902 - fba.levenshtein - INFO - Read pairs processed: 10,000,000
2022-03-15 23:51:53,692 - fba.levenshtein - INFO - Read pairs processed: 20,000,000
2022-03-15 23:56:13,885 - fba.levenshtein - INFO - Read pairs processed: 30,000,000
2022-03-16 00:00:31,902 - fba.levenshtein - INFO - Read pairs processed: 40,000,000
2022-03-16 00:04:52,531 - fba.levenshtein - INFO - Read pairs processed: 50,000,000
2022-03-16 00:06:37,721 - fba.levenshtein - INFO - Number of read pairs processed: 54,024,324
2022-03-16 00:06:37,722 - fba.levenshtein - INFO - Number of read pairs w/ valid barcodes: 27,719,537
2022-03-16 00:06:37,734 - fba.__main__ - INFO - Done.

Matrix generation

Only fragments with correctly matched cell and feature barcodes are included. Use -ul to set the UMI length (default 12). Setting to 0 means no UMIs and read counts are summarized instead. Use -cb_rc to reverse-complement cell barcode sequences in the output matrix if needed.

$ fba count \
    -i feature_barcoding_output_ADT.tsv.gz \
    -o matrix_featurecount_ADT.csv.gz \
    -ul 0

Result summary.

The median number of reads per cell of this ADT library is 2,645.0.

2022-03-16 00:14:03,746 - fba.__main__ - INFO - fba version: 0.0.x
2022-03-16 00:14:03,746 - fba.__main__ - INFO - Initiating logging ...
2022-03-16 00:14:03,746 - fba.__main__ - INFO - Python version: 3.10
2022-03-16 00:14:03,746 - fba.__main__ - INFO - Using count subcommand ...
2022-03-16 00:14:05,873 - fba.count - INFO - UMI-tools version: 1.1.2
2022-03-16 00:14:05,881 - fba.count - INFO - UMI length set to 0, ignoring UMI information. Skipping arguments: "-us/--umi_start".
2022-03-16 00:14:05,881 - fba.count - INFO - Header: read1_seq cell_barcode cb_num_mismatches read2_seq feature_barcode fb_num_mismatches
2022-03-16 00:14:50,518 - fba.count - INFO - Number of read pairs processed: 27,719,537
2022-03-16 00:14:50,549 - fba.count - INFO - Number of cell barcodes detected: 9,151
2022-03-16 00:14:50,549 - fba.count - INFO - Number of features detected: 37
2022-03-16 00:14:50,549 - fba.count - INFO - Counting ...
2022-03-16 00:14:50,901 - fba.count - INFO - Total reads: 27,719,537
2022-03-16 00:14:50,903 - fba.count - INFO - Median number of reads per cell: 2,645.0
2022-03-16 00:14:51,456 - fba.__main__ - INFO - Done.

On average, approximately 25 ADTs (proteins) are detected per cell.

In [1]: import pandas as pd

In [2]: m = pd.read_csv("matrix_featurecount.csv.gz", index_col=0)

In [3]: m.shape
Out[3]: (37, 9151)

In [4]: print(m.sum(axis=1).sort_values(ascending=False).to_string())

RPA-T4_TGTTCCCGCTCAACT               4373383
BC96_TTTGTCCTGTACGCC                 3770330
CD7-6B7_TGGATTCCCGGACTT              3656097
FN50_GTCTCTTGGCTTAAA                 2477291
BJ18_AATCCTTCCGAATGT                 2448859
2D1_TCCCTTGCGATTTAC                  2192495
C398.4A_CGCGCACCCATTAAA              2186772
HIT2_TGTACCCGCTTGTGA                 1864653
UCHT1_CTCATTGTAACTCCT                1243612
Ber-ACT35_(ACT35)_AACCCACCGTTGTTA     666300
DX2_CCAGCTCATTAGAGC                   606868
EH12.2H7_ACAGCGCCGTATTTA              510931
O323_GCACTCCTGCATGTA                  396037
108-17_ACCTTTCGACACTCG                299575
F38-2E2_TGTCCTACCCAACTT               230863
WM59_ACCTTTATGCCACGG                  123346
A1_TTACCTGGTATCCGT                    117428
UCHL1_CTCCGAATCATGTTG                 111140
4B4-1_CAGTAAGTTCGGGAC                  90803
DREG-56_GTCCCTGCAACTTGA                63105
G043H7_AGTTCAGTCAACCGA                 55890
S-HCL-3_TACGCCTATAACTTG                50507
IP26_CGTAACGTAGAGCGA                   49413
CD28.2_TGAGAACGACCCTAA                 27709
HI100_TCAATCCTTCCGCTT                  26717
SK1_GCGCAACTTGATGAT                    12505
A019D5_GTGTGTTGTCCTATG                 10286
HIB19_CTGGGCAATTACTCG                   8570
5.1H11_TCCTTTCCTGATAGG                  8190
A15153G_TTGCTTACCGCCAGA                 7384
HP-3G10_GTACGCAGTCCTTCT                 7150
3G8_AAGTTCACTCTTTGC                     6133
M5E2_TCTCAGACCTCCGTA                    6059
AD2_CAGTTCCTCAGTTCG                     5723
J252D4_AATTCAACCGTCGCC                  4252
MOPC-21_GCCGGACGACATTAA                 1807
QA17A04_AACTCCCTATGGAGG                 1354

In [5]: np.median(m.sum(axis=0))
Out[5]: 2645.0

In [6]: np.median((m > 0).sum(axis=0))
Out[6]: 25.0