v4.3.9

Highlights of the 4.3.9 release:
This release includes an updated version of the README, which informs the user that the Google Drive folder containing the Assignment Sheet templates also now contains a SAMPLE Assignment Sheet denoting the required controls and panel-specific formatting.

v4.3.8

Official 4.3.4 Release Notes
Highlights of the 4.3.4 release:
This release introduces several key updates to the quality control (QC) tests and output files, specifically to ensure that the flags from the QC tests are easily interpretable when reviewing the output files. The goal of these updates is to improve data readability in the Results files, allowing for more effective quality control checks and better-informed decision-making.

The results spreadsheets (found in the RESULTS sub-folder in pipeline outputs) now contains a single Excel file with five sheets containing the following results: ‘CARMEN_Hit_Results’, ‘NTC_Normalized_Quant_Results’, ‘Summary_of_Positive_Samples’, ‘NTC_thresholds’, and ‘Binary_Results’. Specific flags for quality control have been incorporated into these results files: * denote invalid assays, ** denote invalid samples, *** denote samples which test negative for the human internal control RNaseP assay, and † denote No Target Control samples which are removed from the analysis due to appearing as potentially contaminated.

Assays are invalidated based on failing the CPC QC Test (#3). CPC (Combined Positive Control) failure testing has been improved to accommodate testing of RVP, BBP Panel #1, and BBP Panel #2 on the same chip. Users are now asked to denote “_RVP”, “_P1”, or “_P2” to the suffix of each assay and each CPC sample in the Assignment Sheet.

Samples are invalidated based on testing positive for the no-crRNA (negative control) assay.

The NTC Normalized heatmap has been enhanced for better visibility and also contains QC flags, NTC contamination, invalid assays, and invalid samples.

Full list of changes:
RESULTS_{IFC Barcode #}.xlsx:
A single Excel spreadsheet called RESULTS_{IFC Barcode #}.xlsx contains 5 sheets called ‘CARMEN_Hit_Results’, ‘NTC_Normalized_Quant_Results’, ‘Summary_of_Positive_Samples’, ‘NTC_thresholds’, and ‘Binary_Results’. The sheet ‘CARMEN_Hit_Results’ now is color-coded, where negative results are in GREEN and positive results are both in RED and bold. The sheets ‘NTC_Normalized_Quant_Results’ and ‘Binary_Results’ show the results for the positive samples both in RED and bold.

NTC Normalized Heatmap Updates:
Gridlines have been added to the heatmap between samples for improved visibility. Given the improved CPC test (described below), results for each CPC sample are plotted only for their relevant panel (i.e. Results for CPC sample for BBP Panel #1 are only plotted for BBP Panel #1 assays).

NTC Contamination Handling:
A detailed explanation of what happens when NTCs are contaminated has been included in the Quality Control Report. If an NTC probe signal exceeds 0.5 au, it is considered contaminated and removed from the analysis. Contaminated NTCs are marked with hashes on the heatmap. If all NTCs in a run are contaminated, the last NTC’s signal is replaced with 0.5 au and marked with an asterisk.
The files affected by this edit are listed below. These files have been modified to include a dagger symbol (†) indicating that the NTC sample was removed due to contamination, with an explanation below.

• ‘CARMEN_Hit_Results’
• ‘NTC_Normalized_Quant_Results’
• ‘Binary_Results’

CPC Failures:
Modifications made to ensure that the CPC validity check works when running multiple panels on the same chip. The CPC test now checks each panel-specific CPC if users have labeled them as CPC_RVP, CPC_P1, and CPC_P2 in their Assignment Sheet. This ensures that the CPC test works as expected even when running multiple panels simultaneously on a chip. If an assay fails the CPC check, it is considered invalid marked with a single asterisk () followed by an explanation below.
The files affected by this edit are listed below. These files have been modified such that invalid assays that fail the CPC check are marked with single asterisk () followed by an explanation below.

• ‘CARMEN_Hit_Results’
• ‘NTC_Normalized_Quant_Results’
• ‘Summary_of_Positive_Samples’
• ‘NTC_thresholds’
• ‘Binary_Results’

No-crRNA Check:
This is a new test added to assess sample validity. Thus, a column called “Sample Valid? Y/N” to the relevant Results files indicating whether a sample has passed or failed the no-crRNA test. A sample is considered invalid if it has tested negative for the included negative control no-crRNA assay.
The files affected by this edit are listed below. These files have been modified such that invalid samples are marked with triple asterisks (***) with a No in the “Sample Valid? Y/N” column, followed by an explanation below.

• ‘CARMEN_Hit_Results’
• ‘NTC_Normalized_Quant_Results’
• ‘Summary_of_Positive_Samples’
• ‘Binary_Results’

RNaseP Check:
Samples that are negative for the human internal control RNaseP are now flagged with two asterisks (**) followed by an explanation below. Users are directed to read the Quality Control Report.
The files affected by this edit are listed below.

• ‘CARMEN_Hit_Results’
• ‘NTC_Normalized_Quant_Results’
• ‘Summary_of_Positive_Samples’
• ‘Binary_Results’

NTC Thresholds for Normalized Results:
The pipeline now outputs both NTC threshold and NTC normalized thresholds, which are reflected in the sheet “NTC Thresholds” in the RESULT Excel file.

v4.1.0

Highlights of the 4.1.0 release:
Previously, the 3_SD threshold did not account for potential NTC contamination. In this release, this issue has been resolved and the standard deviation is calculated, per assay, for only the NTCs that have passed the contamination check.

Previously, the Negative & Positive Quality Control Checks produced empty csv files as the output upon the absence of sample or assay flags. In this release, in the absence of a sample or assay-level flag, the Check produces a csv file containing a message informing the users of this conclusion.

Previously, the outputs folder contained all outputs in one location. In this release, the outputs folder has been re-organized into three sub-folders - RESULTS_{IFC_barcode}, R&D_{IFC_barcode}, and QUALITY_CONTROL_{IFC_barcode). The user can explore the desired output based on their analysis needs.

Full list of changes:

• Fixed input to threshold.py to be assigned_signal_norm_with_NTC_check.csv
• Built Assay-Level QC Test Explanation file
• Built PDF generator when running the quality control fags for the Positive and Negative Controls

v3.3.1

Highlights of the 3.3.1 release:

Previously, there was an error in the coinf_check module in the qual_checks.py file which appeared when no potentially co-infected samples were flagged. This bug has now been resolved.

v3.3.0

Highlights of the 3.3.0 release:

Previously, the quality control flags produced a single text file that described the samples which had failed to test positive for RNaseP, the assays which had failed to test positive for the CPC positive control, and the assays which had failed to test negative for the NTC and NDC negative controls. In this release, the quality control flags have been converted into tabular outputs in the form of four additional csv files that contain the list of flagged samples (for the RNaseP evaluation) and flagged assays (for the CPC, NDC, and NTC evaluations). A text file describing the results and their initial interpretation is still produced. This modification enables streamlined comparison of results across experiments to fortify diagnostic surveillance in point-of-need capacities.

v3.2.3

Highlights of the 3.2.3 Version

This update includes enhancement of the user interface, improved readability of the outputs, and additional visualizations of the normalized t13 data.

v3.2.2

Highlights of the 3.2.2 Release

This release includes a minor update to software version 3.2.1. The analyze_run.py file has been updated with a correction made to the Qual_Ctrl_Checks module.

v3.2.1

Highlights of the 3.2.1 release:

This release includes a minor update to software version 3.2.0. The ReadMe file has been updated to include a complete description of output files listed as most relevant to those interested in field-based diagnostic surveillance.

v3.2.0

Highlights of 3.2.0 Release

1. Previously, the software mapped assay labels to the heatmap in a randomized order. This has now been corrected such that assay labels in the heatmap layout match the order of input assays in the assignment sheet.

2. Previously, heatmaps were generated for experiments run with 192 samples and 24 viral assays as one singular panel. However, due to difficulty in reading this figure easily, the code now produces a 2 by 1 figure where 96 samples are displayed in a heatmap in the upper sub-figure and 96 samples are displayed in a heatmap in the lower sub-figure.

v3.1.0

Highlights of the 3.1.0 release:

1. We have now included four quality control tests to evaluate assay performance in each experiment that is completed and analyzed. The first three use NTC (No Target Control) negativity, NDC (No Detection Control) negativity, and CPC (Combined Positive Control) positivity to assess each assay based on a scoring system from 0 to 1. The fourth test assesses the performance of the RNaseP (positive control used for human clinical samples) assay based on a scoring system from 0 to 1, generated by the positivity rate of clinical samples for the RNaseP assay. The results are in Assay_Level_QC_Metrics_{barcode_assignment}.csv with the explanation of the four tests in Assay_Performance__QC_Tests_{barcode_assignment}.txt.

2. In addition to the t13_hit_output_{barcode_assignment}.csv file, the output folder also includes t13_hit_binary_output_{barcode_assignment}.csv. This file contains binary 0 and 1 results denoting negative and positive results, respectively.

3. Previously, the Quality Control Checks conducted four checks for NTC negativity, NDC negativity, CPC positivity, and RNaseP evaluation. We have now also included a preliminary Co-Infection assessment that flags any samples that are positive for at least two assays, excluding RNaseP. This assessment has been added to the Quality_Control_Checks_{barcode_assignment}.txt.

4. We have refined the threshold calculations by which samples are denoted positive. Now, the system first checks if any NTC sample may have high contamination (i.e. its fluorescence signal is greater than 0.5 au) for each of the assays tested. If the NTC for an assay is flagged for potential contamination, the NTC is eliminated from the threshold calculation. If all NTCs for a given assay are flagged for contamination, the program will assign keep all but one of the NTC samples and assign it a fluorescence signal of 0.5.

5. Previously, in Quality_Control_Checks_{barcode_assignment}.txt, the NTC check incorrectly suggested that the range of typical fluorescence for an NTC sample is 0.1 – 0.4 au. It has been corrected to the range of 0.1 – 0.5 au.