Output Metrics and Associated Problems

This topic describes possible problems and recommended solutions for sequencing analysis issues. Issues with sequencing metrics might be related to issues that can be resolved in the experimental workflow.

Percentage reads with cell label (Pct_CellLabel-UMI) and percentage aligned uniquely

(Pct_CellLabel_UMI_Aligned_Uniquely) are both low

Possible causes	Recommended solutions
Low sequencing quality	Ensure that the appropriate PhiX % is used for the type of sequencer used. Ensure that the Illumina sequencing flow cell is not over-clustered. Repeat the sequencing run if sequencing quality is suspected to be the reason.
Low library quality	Ensure that the correct panel is used to amplify the sample and the correct amplification protocol and PCR product purification protocols are used. Repeat amplification from leftover PCR1 products, if necessary.

High percentage reads with cell label (Pct_CellLabel-UMI) but low percentage aligned uniquely

(Pct_CellLabel_UMI_Aligned_Uniquely)

Possible causes	Recommended solutions
Incorrect FASTA file panel used for mapping	If <50% alignment, then the wrong panel was likely used. Verify that the correct panel reference file was used.
Incorrect number of sequencing cycles	Run at least 75 x 2 sequencing cycles. The total length of both reads must be at least 102 bp.
Low sequencing quality	Rerun sequencing, and use at least the minimum recommended concentration of PhiX.

Low percentage reads from putative cells

Possible causes	Recommended solutions
Some cells in the samples are not well represented by the panel. Their associated cell labels have very few detectable molecules, so they are classified as noise cell labels	Ensure that the panel matches the sample and species. Ensure that the panel of genes provides good representation across the cells in the sample tested if all cells are to be detected.
Lysis time too long	Ensure that lysis time is exactly 2 minutes and lysis buffer is cold.
Automated pipette settings are incorrect	Ensure that the correct setting is used for the specific step in the cartridge workflow.
Wrong buffer used for bead retrieval from the cartridge	Use only lysis buffer, as indicated in the protocol for bead retrieval.
Mixed species in experiment	Ensure that the panel used contains genes that cover both species.
Excessive dead or dying cells	Proceed with the experiment if cell viability is ≥50%.
Very low bead loading density. The bead loading efficiency on the BD Rhapsody^TM Scanner likely reported failed.	See bead loading density troubleshooting in the BD Rhapsody^TM Single-Cell Analysis System Instrument User Guide (23-21336) or the BD Rhapsody^TM Express Single-Cell Analysis System Instrument User Guide (23-21332).

Number of cells detected in sequencing is much lower or higher than the expected cell number based on imaging

results

Possible causes	Recommended solutions
Wrong inflection point chosen on cell calling graph	Some cell samples or noise profiles create multiple inflection points on the cell calling second-derivitive curve. Guide the basic cell calling algorithm to choose the correct inflection point by running the pipeline with the `Expected_Cell_Count` parameter. Usually this can be the number of cells loaded into the Rhapsody cartridge.
For Targeted mRNA assays: Some cells in the samples are not well represented by the panel. Their associated cell labels have very few detectable molecules, so they are classified as noise cell labels.	If all of the cells are to be detected, ensure that the panel of genes provides good representation across the cells in the sample tested. Ensure that the panel matches the sample and species. If there is more than one bioproduct type in libraries, use another Putative Cell Calling option to troubleshoot.
Cell Capture Beads settled to the bottom of the tube before the start of PCR1.	Ensure that Cell Capture Beads are well suspended just before starting PCR1, and the thermal cycler lid is pre-heated when the PCR tubes are placed on the thermal cycler.
Cell Capture Beads are lost during handling after cartridge use.	Ensure maximum recovery of Cell Capture Beads by using low retention tips and tubes. See product information in the BD Rhapsody^TM Single-Cell Analysis System Instrument User Guide (23-21336) or the BD Rhapsody^TM Express Single-Cell Analysis System Instrument User Guide (23-21332).

Batch effects across multiple libraries

Possible causes	Recommended solutions
Variations in sequencing depth	For Targeted mRNA assays: Examine the status of each bioproduct in `[sample_name]_Bioproduct_Stats.csv` across samples. If there are highly abundant genes with a pass status in one library but a low depth status in another, consider using `[sample_name]_RSEC_MolsPerCell.csv` for analysis. Or, use `[sample_name]_DBEC_MolsPerCell.csv` for analysis after removal of genes that do not have pass status in any of the libraries under consideration.
Variations in cell sample handling protocol	Use a similar cell sample handling protocol for all samples to be analyzed together, noting that temperature, duration of handling, and handling method can affect bioproduct expression.
Differences in thermal cycling	For samples to be analyzed together, it is recommended to perform the PCR amplification of the Cell Capture Beads of those samples in parallel.
Low sequencing depth	For Targeted mRNA assays: Use `[sample_name]_RSEC_MolsPerCell.csv` or use `[sample_name]_DBEC_MolsPerCell.csv` after removal of genes that do not have pass status.