The NIA CARD Long-Read Sequencing group sequences some 30-50 human genomes per week from brain and blood genomic DNA. Given these samples both often belong to larger cohorts (more than 30-50 samples), batch to batch variation is inevitable, and Oxford Nanopore Technologies has steadily modified sequencing library chemistry, flow cells, instrument software, and basecalling models, regular sequencing quality control is critical to examine how our sequencing efforts change over time. The included Python scripts automate the process of collecting key sequencing statistics from weekly and cohort-wide sequencing runs and generate QC analytics as an Excel spreadsheet. They take run output JSON files as input and pull sequencing statistics from exact locations in each JSON hierarchical data structure. The scripts have been written to handle JSON files from MinKNOW version 24.02.19 and earlier. Please report any bugs in the Issues tab to further improve the parser.
CARDlongread_extract_from_json.py takes a list of Oxford Nanopore sequencing report JSON files as inputs (or a directory containing all JSON files to analyze) and returns a table with the following fields per JSON:
Experiment Name, Sample Name, Run Date, PROM ID, Flow Cell ID, Data output (Gb), N50 (kb), MinKNOW Version, Passed Modal Q Score, Failed Modal Q Score, Starting Active Pores, Second Pore Count
Below is sample output from the script:
Experiment Name Sample Name Run Date PROM ID Flow Cell ID Data output (Gb) N50 (kb) MinKNOW Version Passed Modal Q Score Failed Modal Q Score Starting Active Pores
Chile_404 Chile_404 2024-05-14 PC24B302 PAW33034 141.083 23.39 24.02.10 NA NA 7465
Chile_406 Chile_406 2024-05-01 PC24B302 PAW73369 131.699 22.79 24.02.10 NA NA 7339
Chile_509 Chile_509 2024-05-14 PC24B302 PAW61512 117.861 17.12 24.02.10 NA NA 7515
Chile_511 Chile_511 2024-05-08 PC24B302 PAW71368 133.097 20.69 24.02.10 NA NA 7226
Chile_516 Chile_516 2024-05-01 PC24B302 PAW71977 129.544 23.63 24.02.10 NA NA 7516
The file list should contain paths to each report on each line, like so:
/data/CARD_AUX/LRS_temp/CHILE/SEQ_REPORTS/Chile_404/PAW33034/other_reports_PAW33034/report_PAW33034_20240514_2123_c7d4ac03.json
/data/CARD_AUX/LRS_temp/CHILE/SEQ_REPORTS/Chile_406/PAW73369/other_reports_PAW73369/report_PAW73369_20240501_1949_4e83275d.json
/data/CARD_AUX/LRS_temp/CHILE/SEQ_REPORTS/Chile_509/PAW61512/other_reports_PAW61512/report_PAW61512_20240514_2128_5a659d71.json
/data/CARD_AUX/LRS_temp/CHILE/SEQ_REPORTS/Chile_511/PAW71368/other_reports_PAW71368/report_PAW71368_20240508_2046_20d811c8.json
/data/CARD_AUX/LRS_temp/CHILE/SEQ_REPORTS/Chile_516/PAW71977/other_reports_PAW71977/report_PAW71977_20240501_1947_c6a80210.json
Example usage (python CARDlongread_extract_from_json.py -h):
usage: CARDlongread_extract_from_json.py [-h] [--json_dir JSON_DIR] [--filelist FILELIST] [--output OUTPUT_FILE]
Extract data from long read JSON report
optional arguments:
-h, --help show this help message and exit
--json_dir JSON_DIR path to directory containing JSON files, if converting whole directory
--filelist FILELIST text file containing list of all JSON reports to parse
--output OUTPUT_FILE Output long read JSON report summary table in tab-delimited format
CARDlongread_extract_summary_statistics.py then generates a spreadsheet from the output table of CARDlongread_extract_from_json.py containing a sequencing statistics summary table and both violin plot and scatter plot visualizations of data output, read N50, and starting active pores (active pores after starting sequencing). Violin plots are provided separately for output (Gbp) per run (corresponding to each line in the input TSV table), per flow cell, and per experiment. Individual runs (lines in TSV table) are highlighted indicating whether they are an initial run, top up, reconnection, or recovery.
Sequencing runs are typically conducted over 72 hours, with one 20 fmol library load every 24 hours.
An initial run corresponds either to a sequencing run that went through three full loads successfully (one every 24 hours) or to the first part of a full sequencing run where the flow cell was later reconnected at a distinct position (e.g., 1E to 3C) or temporarily disconnected and reconnected at the same position (e.g., 1E).
A reconnection is the second part of a run after a flow cell is disconnected and reconnected, as described above. Reconnections are identified from lines in the JSON parser output TSV that have the same sample name and same flow cell ID.
A recovery run is conducted when either insufficient library is prepared for three full loads (over three days) or when active pores substantially decrease from pre-sequencing checks after the first load. Library is recovered by slow aspiration from the flow cell sample port itself. Library is generally recovered after the first load (often due to active pore drop off) and either loaded onto a new flow cell or to the same flow cell for a subsequent load.
A top up run is conducted on a new flow cell when an experiment has not yielded the desired 90 Gbp (30x coverage) from the initial run (and reconnections/recoveries where applicable). Top up runs are conducted either with initial library if there is sufficient library for more than three loads or with new library if not.
Top up runs are labeled with the suffix _topup and recovery runs are labeled with the suffix _recovery in the sample name column.
Example data visualizations corresponding to data snippets shown earlier:
Total per flow cell output violin plot with embedded box plot and displayed data points:
Per run data output vs. starting active pore scatter plot with run type annotated by color and cutoffs provided for starting active pores (red - less than 5000 pores for ONT warranty return, green - 6500 pores or higher for internal QC) and data output (gray - desired 30X human genome coverage or 90 Gbp sequencing output).
Example usage (python CARDlongread_extract_summary_statistics.py -h):
usage: CARDlongread_extract_summary_statistics.py [-h] [-input INPUT_FILE] [-output OUTPUT_FILE] [-plot_title PLOT_TITLE] [--plot_cutoff | --no-plot_cutoff] [-run_cutoff RUN_CUTOFF]
This program gets summary statistics from long read sequencing report data.
optional arguments:
-h, --help show this help message and exit
-input INPUT_FILE Input tab-delimited tsv file containing features extracted from long read sequencing reports.
-output OUTPUT_FILE Output long read sequencing summary statistics XLSX
-plot_title PLOT_TITLE
Title for each plot in output XLSX (optional)
--plot_cutoff, --no-plot_cutoff
Include cutoff lines in violin plots (optional; default true; --no-plot_cutoff to override) (default: True)
-run_cutoff RUN_CUTOFF
Minimum data output per flow cell run to include (optional, 1 Gb default)
To clone from GitHub and do a test run:
# Download this repo
git clone https://github.com/molleraj/CARDlongread-report-parser.git
cd CARDlongread-report-parser
# Test on pregenerated set of 50 random PPMI JSON files (use exact file provided)
# Random JSON file set generated like so with Linux coreutils shuf (shuffle) command
# shuf -n 50 full_PPMI_json_report_list_090524.txt > random_50_test_PPMI_json_paths_090524.txt
# random_50_test_PPMI_json_paths_090524.txt included in repository as example_json_reports.txt
# Execute with file list of json reports (one per line):
python3 CARDlongread_extract_from_json.py --filelist example_json_reports.txt --output example_output.tsv
# Alternatively, execute on all html files within a directory
# (does not descend into subdirectories)
python3 CARDlongread_extract_from_json.py --json_dir /data/CARDPB/data/PPMI/SEQ_REPORTS/example_json_reports/ --output example_output.tsv
# Make sequencing QC analytics spreadsheet from above QC output table (example_output.tsv)
python3 CARDlongread_extract_summary_statistics.py -input example_output.tsv -output example_summary_spreadsheet.xlsx -plot_title "PPMI tutorial example"Example sequencing QC visualizations from tutorial summary spreadsheet:
Per run output violin plot with embedded box plot and displayed data points, plus run type annotated by point color and red cutoff line for desired 30x coverage/90 Gbp output:
Per run starting active pores violin plot with embedded box plot and displayed data points, plus run type annotated by point color and red cutoff line for recommended minimum 6500 starting active pores:
Per run data output vs. starting active pore scatter plot with run type annotated by point color and cutoffs provided for starting active pores (red - less than 5000 active pores for ONT warranty return, green - 6500 active pores or higher for internal QC) and data output (gray - desired output of 30X human genome coverage or 90 Gbp sequencing output).
