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| There are only four reverse primers (four indexes) provided in the kit. Can I use more than four reverse index primers? | Yes, more than four indexes can be used at a time. The additional primers can be ordered through the IDT TruGrade service and are HPLC purified and resuspended in IDTE pH 8.0 at a 10 μM concentration. See technical bulletin Ordering Additional Indexes for the BD Rhapsody™ Library Reagent Kits for more information. |
| Can the BD Pharmingen™ Stain Buffer be substituted? | The BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) is the only recommended stain buffer. All other buffers have not been tested. |
| Can cells be loaded with media instead of sample buffer? | Cells can be loaded in PBS plus up to 1% BSA, but a large volume of old cell media can have a negative impact on bead-loading efficiency. |
| Can fixed cells be used? | We do not support a protocol for fixed cells at the moment. However, there have been publications on single-cell mRNA applications with methanol fixed cells (https://bmcbiol.biomedcentral.com/articles/10.1186/s12915-017-0383-5#Sec2). Another fixative that preserves mRNA and has been shown to work with other single-cell platforms is CellCover. These methods may work with the BD Rhapsody™ System. Note that calcein only stains live cells. For visualization of fixed cells on the BD Rhapsody™ Scanner, you can try green nuclei stain or strong green stain for dead or fixed cells (https://www.thermofisher.com/order/catalog/product/L23101). We do not anticipate FFPE and formaldehyde fixed cells to work with the BD Rhapsody™ System as mRNA are heavily fragmented by such fixation methods. |
| How stable is the BD Rhapsody™ Cartridge after it has been removed from the foil package (before priming step)? | The BD Rhapsody™ Cartridge is stable for 24 hours after removal from the foil package. |
| How critical is it to keep lysis time to 2 minutes? | It is very critical since longer lysis times can lead to mRNA cross-hybridization between cells (indicated by low % reads to putative cells sequencing metric). |
| How do we calibrate automated pipettes? | Pipettes can be sent to Gilson directly for calibration. Additional information: Lead time is 3–5 days depending on the facility. Yearly service and calibration are recommended. |
| How can we run multiple cartridges in parallel? | The BD Rhapsody™ Single-Cell Analysis System Instrument User Guide provides a recommended protocol for running two cartridges side by side with one sample loading station. |
| How are doublets identified? | Cells are stained with Calcein AM. Individual cells are identified based on fluorescent imaging, and doublets or multiplets that are clustered together are detected based on the shape or eccentricity and variation in fluorescence intensity within the cluster. Doublets in wells are determined by overlaying fluorescent imaging of live cells and brightfield imaging of the wells in the cartridge. |
| How does bead subsampling work? | Beads with synthesized cDNA can be aliquoted as needed and amplified to create a sequencing library. For instance, if the cartridge captures 10,000 cells, proceeds to cDNA synthesis and stores the beads, you can take 10% of the stored beads to make a sequencing library representing ~1,000 cells. |
| If the diversity of UMI is only ~65,000, does it mean you can only capture a total of ~65,000 molecules? | No, the diversity of UMI applies to a gene of a cell not for all genes across a cell and not for all genes across all cells. The definition of a molecule is a unique combination of a UMI sequence, a gene and a cell label. In a typical cell, for a gene, we see a copy number of zero to at most a few thousand. Thus, an UMI diversity of ~65,000 is more than sufficient to ensure each molecule is tagged uniquely. |
| Why isn’t the recommended pooling ratio for VDJ libraries proportionate to the recommended reads/cell? | TCR and BCR libraries have longer fragments than targeted mRNA libraries. Longer fragments are more likely to be outcompeted by smaller fragments during clustering, which results in less data output than expected. Our pooling ratio recommendation has been optimized and validated to obtain the recommended data output ratio for each library. |
| Is the targeted mRNA library necessary for the VDJ CDR3 assay? Can I only sequence BCR and TCR libraries? | At minimum, there are three libraries for VDJ CDR3 analysis. Currently, the pipeline requires mRNA reads for cell calling. If an mRNA library is not added, the bioinformatics pipeline will crash at the cell calling step. |
| Can I use panels other than the immune response panel or custom panel with the VDJ protocol? | While the VDJ CDR3 protocol can technically work with any targeted panel, the immune response panel has genes specific for immune cells to aid immune cell calling, a critical feature for VDJ output. Additionally, to work effectively with our CDR3 VDJ assay, custom panels should include immune response panel markers. |
| Can I pool the BD® AbSeq Library with VDJ libraries to sequence them together? | We cannot guarantee an accurate data output ratio when combining the VDJ and AbSeq libraries. We recommend sequencing AbSeq libraries separately in a PE75 cycle sequencing setup as the small AbSeq fragments will outcompete VDJ libraries. If it is necessary to run the AbSeq libraries with the VDJ library, we recommend using a unique indexing reverse primer for the AbSeq libraries so that the AbSeq reads can be trimmed before running the primary analysis pipeline. |
| I only want to check the protein expression level of my samples. I know mRNA will be captured on the bead, but is it necessary to prepare an mRNA library and sequence it as well along with the AbSeq library? | Yes, currently the pipeline requires mRNA reads for cell calling. To ensure that the pipeline can distinguish live cells from dead cells and debris, prepare a shallow (2,000 reads/cell) mRNA library. If an mRNA library is not added, the bioinformatics pipeline will crash at the cell calling step. |
| Can I flow sort my sample before using the BD Rhapsody™ System workflow? | Yes, we recommend co-staining cells with fluorescent and oligo nucleotide conjugated antibodies (BD Rhapsody™ Single-Cell Multiplexing Kit or BD® AbSeq Ab-Oligos) before cell sorting. See technical bulletin Co-staining cells with fluorescent antibodies and BD Rhapsody™ Antibodies for more information. |
| Can I use the same antibody clone during co-staining? | If it is necessary for your experiment, we recommend co-staining at a 1:1 molar ratio. See technical bulletin Co-staining cells with fluorescent antibodies and BD Rhapsody™ Antibodies for more information. |
| What is the concentration of BD® AbSeq Antibodies? | We provide all BD® AbSeq Ab-Oligos in test size. Each test size is 2 μL; however, the concentration of antibodies may vary. The concentration depends on the identity of the marker type, density of the targets, affinity of the clone, and other factors, so it will differ depending on the antibody specificity. The tubes are packaged according to the titration data to ensure optimal signal-to-noise and performance for each clone. |
| Can I use antibodies other than the off-the-shelf BD® AbSeq Reagents? | Yes, with BD® AbSeq Custom Antibodies, we offer custom conjugation of an AbSeq-qualified oligonucleotide barcode to both antibodies provided by customers and for other BD antibody clones. Contact your regional sales representative to start an order. |
| What is the minimum and maximum number of BD® AbSeq Ab-Oligos used in an assay? | There is no minimum BD® AbSeq Antibody requirement for an assay. The maximum number of BD® AbSeq Antibodies that can be used is 100. We offer a protocol that can support pooling of antibodies for up to 100-plex AbSeq experiments. |
| How can I get the .FASTA reference file for my BD® AbSeq Panel? | Refer to the BD Single-Cell Multiomics Analysis Setup User Guide or use our online BD® AbSeq Panel generator. |
| Can we multiplex samples that have a varying number of antibodies per sample? | Yes, internally, we have tested a 10-plex Ab-oligo multiplexed with a 40-plex sample and saw minimal residual jumping. Ensure that each sample is washed well for best results. |
| Is there any cross reactivity between human and mouse clones used in the BD® Single-Cell Multiplexing Kits (SMK)? | The clone in the BD® Human SMK has been reported to cross-react with non-human primates but may react more weakly. It does not cross-react with mouse. The BD® Mouse SMK does not cross-react with other species. |
| For which cell types can I use the BD® Mouse SMK? | You can use the BD® Mouse SMK for all cell types that express CD45. The Sample Tag in the BD® Mouse SMK is conjugated to an anti-mouse CD45, clone 30-F11 antibody. The CD45 antibody is a lymphocyte and leukocyte marker, expressed on cells including T cells, Pro-B cells, B cells, NK cells, eosinophils, macrophages, LT-HSCs, monocytes, innate lymphoid cells, granulocytes, basophils and mast cells. If you are unsure that your sample expresses the BD® Mouse SMK marker, contact us at scomix@bdscomix.bd.com. |
| For which cell types can I use the BD® Human SMK? | The BD® Human SMK marker is a universal marker expressed on a wide range of cell types, including but not limited to PBMCs, MSCs and different cancer cell lines. If you are unsure if your sample expresses the BD® Human SMK marker, contact us at scomix@bdscomix.bd.com. |
| For BD® SMK libraries, why do I need more reads for different types of cells rather than for the same type? | The Sample Tag targets have different expression levels among different cell types. Most of the reads will go to the predominant cell type if there are various cell types within a sample. We have validated that 600 reads/cell should be enough to recover most of the cells within a sample having various cell types. When you are working with samples with one cell type such as cell lines or samples for which the expression level of the Sample Tag target among the different cell types is similar, 120 reads/cell should be enough to recover most of the cells within the sample. |
| If I used two panels on one sample, how can I run the pipeline analysis? | There are two options: you can either use a combined reference file with duplicated reference sequences removed or specify both references in the pipeline run. |
| What is the minimum and maximum distance that the primer can be from the polyA site in the custom panel design? | 150 bp and 1,000 bp for N1. 150 bp and 400 bp for N2. If there is no good priming site found within these ranges, contact us at scomix@bdscomix.bd.com. |
| Do you have options for species other than human and mouse for targeted mRNA sequencing? |
We currently do not have off-the-shelf panel options for species other than human and mouse. However, it is possible to use the BD Rhapsody™ WTA Assay and BD® AbSeq Custom Antibodies on other species. If your species is one of the following, you can perform the WTA assay first and send the WTA data to the BD Rhapsody™ System support team at scomix@bdscomix.bd.com. We can generate a custom panel for these species based on your WTA data:
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| Can I detect gene variants using the BD Rhapsody™ WTA or targeted assays? | It is difficult to detect gene variants using WTA or targeted assays. The BD Rhapsody™ WTA and targeted assays use 3'-end capture technology and sequence a short fragment on the 3'-end of the transcript, which is mostly a non-coding region. |
| How do we address the amplification efficiency difference between primers? |
When designing primers for a panel, our primer design software utilizes important parameters such as secondary structure, GC content and Tm to improve the uniformity of amplification efficiency across primers. Our molecular indexing technology uses the number of molecules captured before amplification instead of the number of reads (after amplification) for gene-expression measurement. This also reduces the difference in amplification efficiency between primers on gene expression profiling. |
| What is a supplemental panel? | Users have a choice to add up to 100 primers to any of the predesigned panels. |
| What is the minimum and the maximum number of primers for a supplemental panel? | Min = 1, Max = 100 |
| What is the minimum and the maximum number of genes required in a custom panel? | Min = 2, Max = 500 |
| If I am working with a species other than mouse or human, where can I download the reference file? | You can download reference FASTA and GTF reference files from Ensembl or NCBI. Please make sure to generate STAR indexed reference before running the pipeline. |
| I want to sequence my BD Rhapsody™ Libraries on a NovaSeq™ Sequencer. Can I use the 100 cycle kit? | Yes, our pipeline can accommodate shorter read lengths for WTA or Targeted assays to take the advantage of the 100 cycle kit on the NovaSeq™ Sequencer. The acceptable criteria for valid R1 and R2 reads is 60 bases for R1, the entirety of which should match the cell barcode and the UMI, and 42 for R2, which contains the cDNA sequences. |
| Are BD Rhapsody™ Libraries compatible with BGI™ sequencing platforms? | Yes, theoretically, Illumina™ libraries can be converted to BGI™ libraries and sequenced on BGI™ sequencing platforms such as the MGISEQ-2000. |
| Can BD Rhapsody™ Libraries be sequenced with other types of libraries on Illumina platforms? | The read-length requirement for BD Rhapsody™ Libraries is PE75. Note the read-length requirement for other libraries to be run together. If mixing with other types of libraries is required, we recommend that the other libraries be of high diversity. If not, increase at least the recommended amount of PhiX for the sequencer used. |
| Can single end sequencing be used for BD Rhapsody™ Libraries, for example, 150 x 1? | No, we only support paired-end sequencing with a minimum 75 bp on both sides. |
| How much sequencing is sufficient? | Sequencing depth is dependent on application. For cell type clustering, shallow sequencing is sufficient. For in-depth analysis, such as comparison across multiple libraries, deep sequencing is recommended. We recommend meeting the requirement for recursive substitution error correction (RSEC), sequencing depth of ≥6 to reach the threshold of sequencing saturation where most molecules of the library have been recovered. For detailed recommendations, refer to the Sequencing Depths Recommendation section in your specific protocol. |
| If I used two panels on one sample, how can I run the pipeline analysis? | There are two options: you can either use a combined reference file with duplicated reference sequences removed or specify both references in the pipeline run. |
| I knocked in some genes to my organism, how can I modify my reference genome to run the pipeline? | The v1.9.1 pipeline now supports supplemental reference FASTA files, which allow alignment to transgenes, like viral RNA or GFP. You can add transgene sequences with sequence headers and sequences to a FASTA file. On Seven Bridges, the FASTA file of the transgene can be uploaded and included as a part of the pipeline under Supplemental Reference. Locally, the FASTA file can be added to file path under Supplemental_Reference to the yml file. |
| Why are there two subsample settings in the pipeline? What is the difference between these two settings? | The input field in Subsample_Settings is used to perform subsampling on total reads including Sample Tag, AbSeq, and mRNA reads, while the one in Multiplex_Options is only for multiplexed samples run and is used when you want to subsample a specific fraction of a Sample Tag library. |
| How can I download the bioinformatics software from Seven Bridges for local use? | Please follow the BD Single-Cell Multiomics Analysis Setup User Guide (Page17) on how to download and install the pipeline for local use. |
| How long does pipeline analysis usually take? | On Seven Bridges Genomics, about 3 hours and 40 minutes for ~165 million reads for mRNA-only runs. |
| What is the computing requirement for running the pipeline locally? | Refer to the BD Single-Cell Multiomics Analysis Setup User Guide. The local install is intended for the bioinformatics core with servers set up. |
| What is meant by sequencing depth? | Reads with the same cell label, same UMI sequence and same gene, are collapsed into a single raw molecule. The number of reads associated with each raw molecule is reported as the raw adjusted sequencing depth. |
| What are RSEC and DBEC? | They are UMI adjustment algorithms to remove the effect of UMI errors on molecule counting. MI errors that are single-base substitution errors are identified and adjusted to the parent MI barcode using RSEC. Other MI errors derived from library preparation steps or sequencing base deletions are later adjusted using distribution-based error correction (DBEC). Refer to the BD Single-Cell Multiomics Bioinformatics Handbook for more information. |
| When should RSEC and DBEC tables be used? |
The usage depends on the sequencing depth. Scenario 1: If the RSEC sequencing depth is very low (<2), it is likely that all genes do not pass threshold for DBEC (check the metric summary .csv for the number of pass genes). If no genes pass, the DBEC table count is the same as RSEC table count; either can be used. However, we recommend using just the RSEC table. Scenario 2: If the RSEC sequencing depth is between 2 and 6, some genes pass threshold for the DBEC and some don't. We recommend using either of the following options: a. Use the DBEC table but remove the genes that are not passing the threshold for cross-sample comparison. b. Use the RSEC table but keep in mind that there is some level of noise molecules. If cross-sample comparison is necessary, consider only samples with similar sequencing depth or else the level of noise molecules can be very different (since the noise-molecule level increases with increasing depth and the noise molecules remain until DBEC is applied). Scenario 3: If RSEC sequencing depth >6 that is, sequencing approaches saturation and most genes pass DBEC, then we recommend using the DBEC table. |
| For targeted assays, why do you recommend a depth of 6 for RSEC? | We recommend meeting the requirement for the RSEC sequencing depth of ≥6 to reach the threshold of sequencing saturation where most molecules of the library have been recovered. |
| How many reads and molecules are removed by RSEC? | RSEC does not filter out reads. The reads get collapsed into the parent molecular index and each contribute to the MI coverage per molecule. RSEC will reduce the number of observed molecular indices by collapsing indices with an edit distance of 1. The number of molecules removed by RSEC are reflected in the UMI_Adjusted_Stat table, on a per gene basis. Compare the counts Raw_Molecules and RSEC_Adjusted_Molecules. |
| How many reads and molecules are removed by DBEC? |
The number of reads removed by DBEC is reflected in the metric Pct_Cellualr_Reads_With_Amplicons_Retained_By_DBEC. DBEC removes molecules with low depth. The number of molecules removed by DBEC is reflected in the UMI_Adjusted_Stat table, on a per-gene basis. Compare the counts Raw_Molecules, RSEC_Adjusted_Molecules and DBEC_Adjusted_Molecules. |
| I got an RSEC depth of <6. What should I do? Should I sequence further? |
This depends on the application. If it is for simple cell type clustering, additional sequencing may not be necessary. If comparison across multiple samples is necessary, one should consider sequencing further to reach RSEC >6. See the following recommendations: Mean RSEC depth <2: No genes pass the threshold for DBEC. Counts are the same as in the RSEC (edit distance corrected) table. This is acceptable for simple cell type clustering, cell type proportion calculation and identifying significantly expressed genes. 2< Mean RSEC depth <6: Due to the difference in PCR efficiency in different primers, some genes will have a higher depth than others, and some will reach the depth required for DBEC and some will not. If the genes with inconsistent MI adjustment status are associated with a large number of reads, it can lead to an apparent batch effect when plotted on tSNE. If comparison across samples is necessary, use the DBEC molecule tables but remove genes that have not passed in any of the samples. Alternatively, you may also use RSEC molecule tables. Mean RSEC depth > 6: Most genes pass (or are not detected) for DBEC and almost 100% of reads are associated with pass genes. It is acceptable to compare expression profiles across samples. The number of molecules detected approaches saturation. Use DBEC molecule tables. |
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