10X single cell library preparation and sequencing

The requirement is for a custom 10X single cell library preparation and sequencing of 16 samples prepared using the “tissue fixation and dissociation for chromium fixed RNA profiling” protocol (CG000553). Approximately, 7,500-12,500 cells are estimated to be recovered from sequencing. Platforms and instrumentation that conform to expected industry standards and practices are acceptable (e.g., Element AVITI sequencer, 10X Genomics Chromium system).

Fast access to sequence data immediately following sequencing and analysis by LIMS or similar system is required.Evaluation Factors: Technical Approach, Past Performance, and price. Evaluation Factors other than price, when combined, are significantly more important than price. Two days added for vendor response.

Questions and Answers: Please advise if the “tissue fixation” process is to be provided by the Navy Lab, or by the supplier. In other words, will you ship flash-frozen tissue, or provide “fixed” tissue samples? Tissue will be fixed and dissociated using the 10X Genomics “Tissue fixation and Dissociation for Chromium Fixed RNA Profiling” (CG000553) and provided to the vendor for QC, Library Prep, and Sequencing. Please confirm that a read depth of 10,000 reads per cell is adequate.

No, sequencing will be completed using standardized methods and platforms with 75bps paired-end reads at 10E5-10E6 raw reads per cell. Alternatively, 100bps pair-end reads at 50E4-50E5 raw reads per cell will suffice. What data delivery is required, i.e. FASTQ files only, or single-cell bioinformatics? If bioinformatic analysis is needed, please define the deliverables. Delivery of FASTQ files only is required for this request. No single-cell bioinformatics analysis is requested here.

However, vendors are welcome to provide the cost of service without and with 20-40 hours of bioinformatic analysis. Please confirm that a read depth of 10,000 reads per cell is adequate. No, sequencing will be completed using standardized methods and platforms with 75bps paired-end reads at 10E5-10E6 raw reads per cell. Alternatively, 100bps pair-end reads at 50E4-50E5 raw reads per cell will suffice.

Firstly, the 10-fold ranges listed will have a significant impact on cost between the low and high end of the range. As such, we respectfully request that a minimum single target number (per cell) be provided so we can quote the sequencing depth properly. Secondly, the ranges listed are well beyond 10x Genomics recommendations. For example, your 50E4 raw reads per cell is 50x higher than what 10x Genomic recommends.

While we will certainly quote whatever the Navy desires, we are also concerned about oversaturation of the single-cell RNA libraries – which could render most of the data redundant.

Answer: The government’s goal is to classify various cell type-transcriptional patterns in a heterogeneous population in which we expect a low number or some more relatively rare cell types. Review of some literature (ref attached) and consultation suggests 50,000-100,000 raw reads per cell are appropriate in this context. This solicitation requires sequencing to be completed using standardized methods and platforms with 75bps paired-end reads at 100,000 reads per cell, minimally. Alternatively, 100bps pair-end reads at 50,000 raw reads per cell will suffice. Offers that do not meet this requirement will not be considered.