FAQs

Frequently Asked Questions

• General Information

  • What is HIVE scRNAseq?

    • HIVE scRNAseq offers a complete solution for single-cell RNA profiling, transforming single-cells to NGS libraries that can be sequenced on Illumina platforms. The HIVE is a portable, hand-held, single-use device that enables gentle capture, easy storage, and scalable processing for the analysis of single-cell samples.
    • A few distinguishing features of the HIVE:
      • Integrated sample storage
      • Decentralized capture and centralized processing
      • Large sample loading volume
      • Fragile cell recovery
      • Flexible and scalable workflow
      • Strong lysis solution

  • What does the product come with?

    All HIVE parts, reagents, protocols, training materials, and software are provided for sample capture, sample processing, and library preparation. One kit includes enough materials to run 8 samples. Standard lab equipment, such as an oven, centrifuge, thermocycler, and nucleic acid fragment analyzer, are not provided.

  • How do I get started with the HIVE?

    • We recommend that first time users start with the HIVE scRNAseq Sample Capture Kit, Processing Kit, and Starter Bundle.
    • The Starter Bundle includes one-time purchases (e.g. closure tool, filter plate adaptor for thermocyclers, vacuum kit, index plate), protocols, training materials, and our BeeNet analysis software for first time users.
    • We recommend using the training materials (Cell Surrogates and Molecular Controls) for all first-time users. These Kits contain enough HIVE parts and reagents for 8 samples or to:
      • Train 1 user, and run 4 samples
      • Train 2 users, and run 2 samples
    • For more information, see Getting Started with the HIVE scRNAseq v1 

  • Where can I download Sample Capture and Processing protocols?

    Please visit our Support page at honeycomb.bio to download written and video protocols.

• Sample Preparation

  • Are there HIVE-specific requirements for my sample prep?

    We recommend whatever sample prep conditions are best suited for your sample type of interest.

  • What do I do if there are clumps in my single-cell suspension?

    • If you are concerned about clumps you can filter your sample through a 70um cell strainer before loading into the HIVE.
    • The wash step after cell-loading will help to remove large clumps and debris that do not fit into the 60um wells of the HIVE.

  • How low can my cell viability be?

    We recommend aiming for a cell viability >90%.

  • Do you have a recommended protocol for removing red blood cells?

    For red blood cell depletion from blood, we recommend using the EasySep™ RBC Depletion Reagent with the EasySep™ Magnet. We recommend processing 0.5 mL of blood at a time and using the magnet 2-3 times to get the best results.

• Sample Capture

  • What is the shelf-life of my HIVE Collectors?

    The HIVE is currently verified for up to 9-month shelf-life at -20C.  Testing of longer shelf-life periods is ongoing.

  • Can I thaw my HIVE Collectors at 4C instead of room temperature prior to cell-loading?

    Yes, thawing at 4C overnight is similar to thawing at room temperate for 30 minutes.

  • How long can my HIVE Collector be at room temperature prior to cell-loading?

    1 hour, move to 4C if longer than 1 hour.

  • My cells were not ready for loading after all, can I re-freeze my thawed HIVE Collector?

    Yes, you can re-freeze HIVE Collectors once to be re-used at another time. Testing of more freeze-thaw cycles is ongoing.

  • How many cells can be loaded per HIVE?

    • We recommend loading 15,000 cells per HIVE.
    • The loading range is 500-30,000 cells per HIVE 
    • When counting, please count the total number of cells- live and dead

  • What is the sample loading volume?

    • We recommend loading 1 mL, with the option to make up the rest of the volume with sample buffer if 1 mL of sample is unavailable. The HIVE can hold up to 4 mL.
    • If your sample volume is greater than 4mL you can load multiple times, sequentially.

  • What sample types can I use?

    HIVE scRNAseq is feasible for eukaryotic cells containing mRNA transcripts with 3’ poly-A tails. We have tested a variety of mammalian cell types and the verified cell lines and primary cells including human filtered blood, PBMCs, and mouse splenocytes.

  • Can I use an automated cell counter instead of the disposable hemocytometer?

    Yes, but we recommend first doing a comparison with the hemocytometer to ensure that your cell counter is accurate.

  • What type of media should I suspend my cells in before loading?

    The HIVE is compatible with a broad range of sample buffers. We recommend ~0.1% protein, but up to 10% protein is okay. An extra wash is recommended for >1% protein.

  • How do you ensure that only one cell is entering each well?

    Cell loading follows a Poisson distribution, so most wells contain a single cell but with increasing cell number, there is an increasing rate of doublet formation.

  • What is the doublet rate?

    The following table gives the theoretical multiplate rate for Poisson loading based on the number of cells that were loaded and the loading efficiency.

    

• Storage & Shipping

  • How long can I store a cell-loaded HIVE at -20C?

    The HIVE is currently verified for up to five months of storage at -20°C, and successfully tested with filtered blood for up to eight months. Testing of longer storage periods is ongoing.

  • Can I store my cell-loaded HIVE at 4C?

    You can store your cell-loaded HIVE for up to 1 week at 4C. Store at -20C if longer than 1 week is needed.

  • Can I store my cell-loaded HIVE at -80C?

    Storing your cell-loaded HIVE at -80C is similar to storage at -20C. Testing of longer storage periods is ongoing.

  • Can I go directly from Sample Capture to Processing without storing cell-loaded HIVEs?

    Yes, if you are not stopping after Sample Capture, let the cell-loaded HIVEs incubate with the Cell Preservation Solution for 30 minutes before starting the Processing protocol.

  • Why do I have to ship cell-loaded HIVEs with dry-ice?

    The Cell Preservation Solution does not fully freeze at -20C.  Cell-loaded HIVEs need to be fully frozen (while flat) for shipment.

  • How should I store cell-loaded HIVEs that were shipped to me?

    After receiving cell-loaded HIVEs that were shipped on dry ice, we recommend storing them at -80C until processing.  For more information, see: QC for cell-loaded HIVEs

• HIVE Processing & Library Prep

  • How many samples can I process at once and how long does it take?

    Our workflow offers a high degree of flexibility, as each sample is independent. Under normal circumstances, 1 to 24 samples can be worked with in parallel and can be processed and ready for sequencing in 1.5 days.

  • How do I know I have successfully recovered the beads from the HIVE?

    After centrifugation, a compact white pellet will be visible at the bottom of the bead collector (more easily seen when holding the spin plate overhead).  While removing 300ul, beads will be present in the pipette tip, and the solution will appear cloudy.  Afterwards, the pellet in the bead collector will no longer be visible.

  • Can I use a circular 96-well plate magnet instead of a bar magnet?

    We recommend a bar magnet for ease of use, but other magnets are acceptable if you’ve successfully used them previously for SPRI clean-up.

  • How do I dispose of my used HIVE parts?

    Used HIVE parts can be disposed as non-sharps in standard bio-safety waste.

  • Can the HIVE lysis solution inactivate infectious disease samples?

    Yes, the lysis solution has been shown to inactivate SARS-CoV-2-infected human whole blood.

    Lysis Solution Data

• QC, Pooling & Sequencing

  • What sequencing platform do you recommend?

    • HIVE scRNAseq libraries are currently compatible with Illumina sequencing platforms capable of custom dual index paired-end sequencing. We recommend NovaSeq, NextSeq 2000, or NextSeq500/550 (NovaSeq v1.5 kits and NextSeq v2.0 (or later) and are compatible).
    • Custom sequencing and index primers are provided with the Processing Kit

  • How many reads are required for each HIVE?

    Required reads can vary depending on number of cells, application, data quality, and sample types. We generally recommend 25,000-50,000 reads per cell.

  • Which sequencing kits do you recommend?

    • NextSeq 500/550 – 75 High Output cycle kit
    • NextSeq 2000- 100 cycle kit
    • NovaSeq – 100 cycle kit

  • What is the recommended sequencing configuration?

    • R1: 25  R2: 50  I1: 8  I2: 8
    • Appendix 2 on page 24 on the HIVE scRNAseq Processing User Protocol goes into detail about setting up for sequencing.

  • How can I check my sequencing quality?

    • Base quality metrics are used to assess the accuracy of sequencing technology. Illumina provides 2 metrics for assessing base quality: %PF (The percentage of clusters that pass Illumina’s preset filter) and %QC30 (The percentage of bases with a quality score of 30 or higher)
    • FastQC can be used to evaluate your sequencing data. FastQC is an open-source quality control tool for high throughput sequence data developed at Babraham Institute. More information and the download can be found at: https://www.bioinformatics.babraham.ac.uk/projects/fastqc/

  • How should I demultiplex my samples?

    If all indexes used were from the same column of the index plate, for example A1-H1, demultiplex samples based on the index 1 read only, since the index 2 reads will be identical. If the indexes used were from multiple columns, demultiplex based on the index 1 and index 2 read.

  • When setting up for sequencing, what fragment size should I use?

    We recommend using the fragment size of 750 bp for calculating the molar concentration of HIVE libraries when loading Illumina sequencers, even if your fragment analyzer shows a different peak size.  Please provide this information to your sequencing facility.

• Software & Analysis

  • What analysis software is provided?

    BeeNet™, our software solution specifically designed for HIVE scRNAseq.

  • How can I access and use the software?

    • Please visit our Support page at honeycomb.bio for instructions on installation and analysis.
    • BeeNet can be downloaded and used on clusters or cloud via Linux command line. We also host BeeNet on Terra for users with no command line experience.

  • Can I download example data?

    Yes, data is available for download here

  • What is the input file for the BeeNet analysis software?

    Demultiplexed Read 1 and Read 2 FASTQ files from Illumina paired-end sequencing.

  • Which reference genome for human do you use?

    We have both hg19 and GRhC38 available for use.

  • Can we use our own references?

    Currently we only support the references we provide. If there is a reference you are interested in using and it is not available via BeeNet, submit a support request at honeycomb.bio and we can work with you to build a new reference file.

  • Do I need to download references every time?

    If you have already downloaded the references and they are still available in the same system, you do not need to download them again.

  • Does the BAM file contain mapping quality information?

    Yes it does, however, our BAM files do not contain molecule count information.

  • Do you annotate intronic regions?

    Intronic regions are annotated in the BAM file, but they are not included in molecule counts.

  • What types of files does BeeNet generate?

    BeeNet produces three types of files: BAM, count matrices, and quality metrics.

  • What does the RCM.tsv.gz file tell me?

    This file displays the number of reads for each unique cell barcode that maps to a specific gene in the reference genome and can be used for down-sampling to see sequencing saturation.

  • What does the TCM.tsv.gz file tell me?

    This file displays the number of unique molecule counts for each transcript that mapped to a specific gene in the reference genome, and can be used for downstream analysis such as Seurat and Scanpy.

  • What does the CMSummary.tsv file tell me?

    This file displays the number of total genes and number of molecule counts for each transcriptome barcode for quick reference as QC.

  • What does the ReadsQC.tsv file tell me?

    This file shows the QC metrics per cell barcode.  It shows the number of filtered out reads, polyA reads, reads with primer sequences, and the number of reads mapped to the genome or annotated exons.  These metrics are specified for each cell barcode, based on the number of barcodes specified in the analysis.

  • What does the SampleQC.tsv file tell me?

    This file shows the QC metrics for all the reads in the fastq files for the sample.  It also provides the number of filtered out poor quality reads, polyA reads, and reads with primer sequences. These metrics are for the sum of all cell barcodes within a fastq file for a given sample.