V2

.gitignore

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+subworkflows/prepare_input_visium_hd.nf
+.claude/

assets/10x_config/adapter_seq_config.csv

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+technology,fwd_primer_f,fwd_primer_r,rev_primer_f,rev_primer_r,TSO_f,TSO_r
+10x3v2,CTACACGACGCTCTTCCGATCT,AGATCGGAAGAGCGTCGTGTAG,CCCATGTACTCTGCGTTGATACCACTGCTT,AAGCAGTGGTATCAACGCAGAGTACATGGG,,
+10x3v3,CTACACGACGCTCTTCCGATCT,AGATCGGAAGAGCGTCGTGTAG,CCCCTCTGCGTTGATACCACTGCTT,AAGCAGTGGTATCAACGCAGAGGGG,,
+10x3v4,CTACACGACGCTCTTCCGATCT,AGATCGGAAGAGCGTCGTGTAG,CCCCTCTGCGTTGATACCACTGCTT,AAGCAGTGGTATCAACGCAGAGGGG,,
+10x5v2,CTACACGACGCTCTTCCGATCT,AGATCGGAAGAGCGTCGTGTAG,GTACTCTGCGTTGATACCACTGCTT,AAGCAGTGGTATCAACGCAGAGTAC,TTTCTTATATGGG,CCCATATAAGAAA
+10x5v3,CTACACGACGCTCTTCCGATCT,AGATCGGAAGAGCGTCGTGTAG,GTACTCTGCGTTGATACCACTGCTT,AAGCAGTGGTATCAACGCAGAGTAC,TTTCTTATATGGG,CCCATATAAGAAA
+visium-v1,CTACACGACGCTCTTCCGATCT,AGATCGGAAGAGCGTCGTGTAG,CCCATGTACTCTGCGTTGATACCACTGCTT,AAGCAGTGGTATCAACGCAGAGTACATGGG,,
+visium-v2,CTACACGACGCTCTTCCGATCT,AGATCGGAAGAGCGTCGTGTAG,CCCATGTACTCTGCGTTGATACCACTGCTT,AAGCAGTGGTATCAACGCAGAGTACATGGG,,
+visium-v3,CTACACGACGCTCTTCCGATCT,AGATCGGAAGAGCGTCGTGTAG,CCCATGTACTCTGCGTTGATACCACTGCTT,AAGCAGTGGTATCAACGCAGAGTACATGGG,,
+visium-v4,CTACACGACGCTCTTCCGATCT,AGATCGGAAGAGCGTCGTGTAG,CCCATGTACTCTGCGTTGATACCACTGCTT,AAGCAGTGGTATCAACGCAGAGTACATGGG,,
+visium-v5,CTACACGACGCTCTTCCGATCT,AGATCGGAAGAGCGTCGTGTAG,CCCATGTACTCTGCGTTGATACCACTGCTT,AAGCAGTGGTATCAACGCAGAGTACATGGG,,

assets/10x_config/barcode_coordinate_config.csv

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+technology,barcode_path,spatial_coordinate_path
+10x3v2,737K-august-2016.txt,
+10x3v3,3M-february-2018_TRU.txt.gz,
+10x3v4,3M-3pgex-may-2023_TRU.txt.gz,
+10x5v2,737K-august-2016.txt,
+10x5v3,3M-5pgex-jan-2023.txt.gz,
+visium-v1,visium-v1.txt,visium-v1_coordinates.txt
+visium-v2,visium-v2.txt,visium-v2_coordinates.txt
+visium-v3,visium-v3.txt,visium-v3_coordinates.txt
+visium-v4,visium-v4.txt,visium-v4_coordinates.txt
+visium-v5,visium-v5.txt,visium-v5_coordinates.txt

assets/10x_config/flank_seq_config.csv

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+technology,left_flank,barcode,umi,right_flank
+10x3v2,CTACACGACGCTCTTCCGATCT,????????????????,??????????,TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
+10x3v3,CTACACGACGCTCTTCCGATCT,????????????????,????????????,TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
+10x3v4,CTACACGACGCTCTTCCGATCT,????????????????,????????????,TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
+10x5v2,CTACACGACGCTCTTCCGATCT,????????????????,??????????,TTTCTTATATGGG
+10x5v3,CTACACGACGCTCTTCCGATCT,????????????????,????????????,TTTCTTATATGGG
+visium-v1,CTACACGACGCTCTTCCGATCT,????????????????,????????????,TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
+visium-v2,CTACACGACGCTCTTCCGATCT,????????????????,????????????,TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
+visium-v3,CTACACGACGCTCTTCCGATCT,????????????????,????????????,TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
+visium-v4,CTACACGACGCTCTTCCGATCT,????????????????,????????????,TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
+visium-v5,CTACACGACGCTCTTCCGATCT,????????????????,????????????,TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT

bin/reverse_complement_fastq.py

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+#!/usr/bin/env python3
+import argparse
+import sys
+
+DNA_COMPLEMENT = str.maketrans("ACGTNacgtn", "TGCANtgcan")
+
+def parse_args():
+    parser = argparse.ArgumentParser(description = "Reverse Complementing FASTQ files generated by Flexiplex")
+    parser.add_argument("-i", "--input", default = '-', help = "Input FASTQ file")
+    parser.add_argument("-o", "--output", default = '-', help = "Output FASTQ file")
+    args = parser.parse_args()
+
+    return args
+
+def modify_read_id(id):
+    """
+    Reverses strand direction tag in the Read ID. Example: GGAATCTCAAGCGCAA_TGGTCTTATTAA#9862034a-576a-44ad-bab9-30e8e9927dde_+1of1 
+    will be modified to GGAATCTCAAGCGCAA_TGGTCTTATTAA#9862034a-576a-44ad-bab9-30e8e9927dde_-1of1
+
+    Args:
+        id (str): Read ID
+
+    Returns:
+        str: Modified Read ID
+    """
+    rev_dict = {'+': '-', '-': '+'}
+    id_list = id.split('_')
+
+    # Exception handling in the event of unusual Flexiplex Read ID format
+    try:
+        strand_dir = id_list[-1][0]
+        id_list[-1] = rev_dict.get(strand_dir, strand_dir) + id_list[-1][1:]
+    except IndexError:
+        pass
+
+    return "_".join(id_list)
+
+def modify_read_description(header):
+    """
+    Reverses strand direction tag in the Description Header. Example: GGAATCTCAAGCGCAA_TGGTCTTATTAA#9862034a-576a-44ad-bab9-30e8e9927dde_+1of1  CB:Z:GGAATCTCAAGCGCAA   UB:Z:TGGTCTTATTAA
+    will be modified to GGAATCTCAAGCGCAA_TGGTCTTATTAA#9862034a-576a-44ad-bab9-30e8e9927dde_-1of1    CB:Z:GGAATCTCAAGCGCAA   UB:Z:TGGTCTTATTAA
+
+    Args:
+        header (str): Description header
+
+    Returns:
+        str: Modified description header
+    """
+
+    header_list = header.split()
+    original_read_id = header_list[0]
+    modified_read_id = modify_read_id(original_read_id)
+
+    # Preserves trailing tags like CB:Z: / UB:Z:
+    return modified_read_id + header[len(original_read_id):]
+
+def reverse_complement_seq(seq):
+    """
+    Reverse complements a DNA sequence
+
+    Args:
+        seq (str): DNA sequence
+
+    Returns:
+        str: Sequence of the reverse complement 
+    """
+    return seq[::-1].translate(DNA_COMPLEMENT)
+
+def reverse_phred_scores(phred_scores):
+    """
+    Reverses Phred Quality Sequence
+
+    Args:
+        phred_scores (str): Phred quality score of the forward strand
+
+    Returns:
+        str: Phred quality score of the reverse complement
+    """
+    return phred_scores[::-1]
+
+if __name__ == "__main__":
+    # Parse arguments
+    args = parse_args()
+    f_in = sys.stdin if args.input == '-' else open(args.input, 'r')
+    f_out = sys.stdout if args.output == '-' else open(args.output, 'w')
+
+    # Track number of reads processed
+    reads_processed = 0
+
+    while True:
+        # Retrieve information for each read (stored in 4 lines)
+        header = f_in.readline().rstrip()
+        # Stop once header is empty
+        if not header: 
+            break
+
+        dna_seq = f_in.readline().rstrip()
+        f_in.readline() # Read separator line but do not store it
+        phred_seq = f_in.readline().rstrip()
+
+        # Get header, DNA sequence and Phred sequence for reverse complement
+        rc_header = modify_read_description(header)
+        rc_dna_seq = reverse_complement_seq(dna_seq)
+        rc_phred_seq = reverse_phred_scores(phred_seq)
+
+        # Write output
+        f_out.write(f"{rc_header}\n{rc_dna_seq}\n+\n{rc_phred_seq}\n")
+
+        # Increment read counter
+        reads_processed += 1
+        if reads_processed % 1000000 == 0:
+            sys.stderr.write(f"\rProcessed {reads_processed/1000000} million reads")
+
+    # Close file handles if not stdin/stdout
+    if args.input != '-': f_in.close()
+    if args.output != '-': f_out.close()

containers/alignment/Dockerfile

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+FROM mambaorg/micromamba:git-c0f93d2-amazon2023
+
+RUN micromamba install -y -n base \
+	-c conda-forge \
+	-c bioconda \
+	minimap2=2.30 \
+	samtools=1.23 \
+	procps-ng \
+&& micromamba clean -ay
+
+ENV PATH=/opt/conda/bin:$PATH

containers/preprocess/Dockerfile

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+FROM mambaorg/micromamba:git-c0f93d2-amazon2023
+
+RUN micromamba install -y -n base \
+	-c conda-forge \
+	-c bioconda \
+	chopper=0.12.0 \
+	flexiplex=1.02.5 \
+	cutadapt=5.2 \
+	pigz=2.8 \
+	procps-ng \
+&& micromamba clean -ay
+
+ENV PATH=/opt/conda/bin:$PATH

containers/r/Dockerfile

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+FROM rocker/r-base:4.4.1
+
+# install system dependencies
+RUN apt-get update && apt-get install -y \
+	  libcurl4-openssl-dev \
+	  procps && rm -rf /var/lib/apt/lists/*
+
+# install Seurat Object (v5.3.0), Seurat (v5.4.0), and Bambu
+RUN R -e "install.packages(c('pak', 'devtools', 'BiocManager'), repos='https://cloud.r-project.org')"
+RUN R -e "pak::pkg_install(c('SeuratObject@5.3.0', 'Seurat@5.4.0', 'GoekeLab/bambu@devel_pre_v4'))"