10x Genomics

Last refreshed at Thu Apr 18 12:27:44 2019.

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Introduction

The NGSC has a 10x Genomics Single Cell Controller (SCC). This controller can perform the single cell RNA-Seq and V(D)J experiments, but not whole genome sequencing, whole exome sequencing, and de novo assembly.

The SCC can prepare up to 8 samples at a time, where a sample is a tube of single cells.

The SCC takes about 10 minutes to perform the initial steps, but then requires about a day and a half of library prep to follow.

The libraries created by the SCC can contain 3500 to 5000 or more cells, however the transcriptome complexity is noticably sparser than is acheived by our Fluidigm C1. However the 10x has a much lower cost per cell.

The purpose of this document is to answer the FAQs for using the 10x at the NGSC. We also provide a set of links to documents at 10x.

How It Works

The 10x merges about 50% of your single cells with droplets that contain a GEM that has one of 700,000 barcodes along with unique molecular identifiers (UMIs). This is the only step that happens on the controller. Following this the droplet emulsion is broken, cDNA is created and amplified. Finally library prep is performed to create Illumina-compatible libraries. Each sample can be assigned barcodes.

V(D)J libraries can be made instead of, or in addition to the 3’ RNA-Seq libraries. If both are required, then an additional round of amplification is needed to create additional material.

The recommended sequencing is geometry is 26x98. The 26bp first read includes the cell barcode and UMI. The 98bp second read contains the cDNA section near the end of the transcript. The cDNA fragments have a range of lengths. The NGSC generally sequences 10x libraries in HiSeq 4000 paired-end runs that are longer than 26x98 in order to avoid delays while providing lower cost sequencing than the NextSeq. The lane is charged as a 75PE lane.

The initial data processing following sequencing is performed by software (cell ranger) that is provided by 10x Genomics. The steps are mkfastq and count. The purpose of mkfastq is clear. However one thing to note is that since we generally sequence more than the minimum number of read and barcode cycles, this step is configured to trim off the excess cycles. Also it requires (at the moment) creating a samplesheet by hand. The second step, count, determines which cell barcodes correspond to cells, as opposed to empty droplets. It then aligns the reads to the genome and produces a report which explains how the ‘empty droplet’ decision was made. The report also includes clustering and an interactive display which can be used to explore the data. Finally count produces a cell x gene file which can be used for further analysis.

Later data analysis can be performed with 10x software Loupe Cell Browser which is a desktop-based software package.

Costs

The cost of an experiment depends on four factors:

  1. What type of libraries are made.
  2. How many samples.
  3. How many batches of samples.
  4. How many cells are captured.

The cost of reagents varies a bit according to the type of library. In particular, making V(D)J libraries adds extra per-sample costs.

Each sample generates costs for reagents, quality checks, and sequencing.

Each batch of samples incurs costs for the 10X chip and labor for library prep, which must be completed soon after cells are captured.

Sequecing is typically done to the recommended depth for the library type. We recommend pooling batches of samples together then sequencing together as deeply as needed. Typically 6000 cells can be sequenced in one HiSeq 4000 lane.

Here are example quotes for a few scenarios.

Minimal Experiment

We have just one sample and capture fewer than 6000 cells so the sequencing is completed in one lane.

The total is $5,571.

. ServiceType ServiceCode ListPrice Count ExtendedPrice Description
1 10XGenomics CSCC_Run $ 440 1 $ 440 A run of the 10x Genomics Chromium Single Cell Controller with between 1 and 8 samples, charged separately
2 10XGenomics CSCC_3pCapture $1,874 1 $1,874 A sample in a run of the 10x Genomics Chromium Single Cell Controller with between 1 and 8 samples.
3 10XGenomics CSCC_3pLibraryPrep $ 122 1 $ 122 Reagents for final library prep of a sample after a successful capture run.
4 QualityCheck qc-Kapa $ 45 1 $ 45 Molarity measurement of library using Kapa Biosystems Library quant kit. 3 technical replicates of 3 dilutions of sample.
5 QualityCheck qc-bioAnalyzer $ 25 2 $ 50 RNA or library quality check with Agilent bioAnalyzer using any of the DNA or RNA chips.
6 QualityCheck qc-qubit $ 5 2 $ 10 RNA or DNA concentration measurement using LifeTech qubit fluorimeter.
7 Labor Hour-Lab $ 100 8 $ 800 15 minutes per pool. Usually 8 to 12 hours per library prep batch.
8 Sequencing sq-075PE-hs4000-Vn $2,228 1 $2,228 Sequencing 150bp from 2 ends of library on HiSeq4000 to get approximately 312 million reads pairs.

Four Samples, One batch, Two Lanes

In this scenario, we capture four samples in one run, and capture about 3000 cells per sample so that only 2 lanes of sequencing are needed.

The total is $15,129.

. ServiceType ServiceCode ListPrice Count ExtendedPrice Description
1 10XGenomics CSCC_Run $ 440 1.00 $ 440 A run of the 10x Genomics Chromium Single Cell Controller with between 1 and 8 samples, charged separately
2 10XGenomics CSCC_3pCapture $1,874 4.00 $7,497 A sample in a run of the 10x Genomics Chromium Single Cell Controller with between 1 and 8 samples.
3 10XGenomics CSCC_3pLibraryPrep $ 122 4.00 $ 488 Reagents for final library prep of a sample after a successful capture run.
4 QualityCheck qc-Kapa $ 45 4.00 $ 180 Molarity measurement of library using Kapa Biosystems Library quant kit. 3 technical replicates of 3 dilutions of sample.
5 QualityCheck qc-Pooling $ 1 1.00 $ 1 Mix samples in equi-molar amounts to ensure as close to even read counts as possible.
6 QualityCheck qc-bioAnalyzer $ 25 8.00 $ 200 RNA or library quality check with Agilent bioAnalyzer using any of the DNA or RNA chips.
7 QualityCheck qc-qubit $ 5 8.00 $ 40 RNA or DNA concentration measurement using LifeTech qubit fluorimeter.
8 Labor Hour-Lab $ 100 8.25 $ 825 15 minutes per pool. Usually 8 to 12 hours per library prep batch.
9 Sequencing sq-075PE-hs4000-Vn $2,228 2.00 $4,457 Sequencing 150bp from 2 ends of library on HiSeq4000 to get approximately 312 million reads pairs.
10 Informatics IT-Analysis $ 391 1.00 $ 391 Higher-level analysis pipeline, e.g., differential expression. CPU and data storage charges extra.
11 Informatics IT-RL $ 305 2.00 $ 610 Basic library-specific data analysis pipeline run on the the data from one lane. CPU and data storage charges are separate.

Four Samples, One batch, Two Lanes With V(D)J

In this scenario, we capture four samples in one run, and capture about 3000 cells per sample so that only 2 lanes of sequencing are needed (like the experiment above), but we also do both B-cell and T-cell V(D)J sequencing. This adds some library prep reagent costs, a 150PE MiSeq run, and a bit more complicated data analysis.

The total is $20,107.

. ServiceType ServiceCode ListPrice Count ExtendedPrice Description
1 10XGenomics CSCC_Run $ 440 1.0 $ 440 A run of the 10x Genomics Chromium Single Cell Controller with between 1 and 8 samples, charged separately
2 10XGenomics CSCC_5pCapture $1,874 4.0 $7,497 A sample in a V(D)J run of the 10x Genomics Chromium Single Cell Controller with between 1 and 8 samples
3 10XGenomics CSCC_5pLibraryPrep $ 208 4.0 $ 830 Reagents for final 5p mRNA-Seq library prep of a sample after a successful capture run.
4 10XGenomics CSCC-VDJ-Human-B-Enrichment $ 43 4.0 $ 171 Enrichment of human B-cell receptor transcripts.
5 10XGenomics CSCC-VDJ-Human-Tcell-Enrichment $ 43 4.0 $ 171 Enrichment of human T-cell receptor transcripts.
6 QualityCheck qc-Kapa $ 45 4.0 $ 180 Molarity measurement of library using Kapa Biosystems Library quant kit. 3 technical replicates of 3 dilutions of sample.
7 QualityCheck qc-Pooling $ 1 2.0 $ 2 Mix samples in equi-molar amounts to ensure as close to even read counts as possible.
8 QualityCheck qc-bioAnalyzer $ 25 24.0 $ 600 RNA or library quality check with Agilent bioAnalyzer using any of the DNA or RNA chips.
9 QualityCheck qc-qubit $ 5 24.0 $ 120 RNA or DNA concentration measurement using LifeTech qubit fluorimeter.
10 Labor Hour-Lab $ 100 12.5 $1,250 15 minutes per pool. Usually 8 to 12 hours per library prep batch.
11 Sequencing sq-075PE-hs4000-Vn $2,228 2.0 $4,457 Sequencing 150bp from 2 ends of library on HiSeq4000 to get approximately 312 million reads pairs.
12 Sequencing sq-150PE-hs4000-Vn $3,083 1.0 $3,083 Sequencing 150bp from 2 ends of library on HiSeq4000 to get approximately 312 million reads pairs.
13 Informatics IT-Analysis $ 391 1.0 $ 391 Higher-level analysis pipeline, e.g., differential expression. CPU and data storage charges extra.
14 Informatics IT-RL $ 305 3.0 $ 916 Basic library-specific data analysis pipeline run on the the data from one lane. CPU and data storage charges are separate.

Eight Samples, Four Batches, Eight lanes

Finally, here is a large set of samples spread out over four 10X runs, e.g., if samples are coming from rare patient samples.

The total is $58,790.

. ServiceType ServiceCode ListPrice Count ExtendedPrice Description
1 10XGenomics CSCC_Run $ 440 4.00 $ 1,758 A run of the 10x Genomics Chromium Single Cell Controller with between 1 and 8 samples, charged separately
2 10XGenomics CSCC_3pCapture $1,874 8.00 $14,994 A sample in a run of the 10x Genomics Chromium Single Cell Controller with between 1 and 8 samples.
3 10XGenomics CSCC_5pCapture $1,874 8.00 $14,994 A sample in a V(D)J run of the 10x Genomics Chromium Single Cell Controller with between 1 and 8 samples
4 10XGenomics CSCC_5pLibraryPrep $ 208 8.00 $ 1,661 Reagents for final 5p mRNA-Seq library prep of a sample after a successful capture run.
5 10XGenomics CSCC-VDJ-Human-B-Enrichment $ 43 8.00 $ 342 Enrichment of human B-cell receptor transcripts.
6 10XGenomics CSCC-VDJ-Human-Tcell-Enrichment $ 43 8.00 $ 342 Enrichment of human T-cell receptor transcripts.
7 QualityCheck qc-Kapa $ 45 24.00 $ 1,080 Molarity measurement of library using Kapa Biosystems Library quant kit. 3 technical replicates of 3 dilutions of sample.
8 QualityCheck qc-Pooling $ 1 3.00 $ 3 Mix samples in equi-molar amounts to ensure as close to even read counts as possible.
9 QualityCheck qc-bioAnalyzer $ 25 48.00 $ 1,200 RNA or library quality check with Agilent bioAnalyzer using any of the DNA or RNA chips.
10 QualityCheck qc-qubit $ 5 48.00 $ 240 RNA or DNA concentration measurement using LifeTech qubit fluorimeter.
11 Labor Hour-Lab $ 100 48.75 $ 4,875 15 minutes per pool. Usually 8 to 12 hours per library prep batch.
12 Sequencing sq-075PE-hs4000-Vn $2,228 4.00 $ 8,913 Sequencing 150bp from 2 ends of library on HiSeq4000 to get approximately 312 million reads pairs.
13 Sequencing sq-150PE-hs4000-Vn $3,083 2.00 $ 6,166 Sequencing 150bp from 2 ends of library on HiSeq4000 to get approximately 312 million reads pairs.
14 Informatics IT-Analysis $ 391 1.00 $ 391 Higher-level analysis pipeline, e.g., differential expression. CPU and data storage charges extra.
15 Informatics IT-RL $ 305 6.00 $ 1,832 Basic library-specific data analysis pipeline run on the the data from one lane. CPU and data storage charges are separate.

FAQs

  • Can I use spike in controls with the 10x? - Generally, no. If a spike in were used, then most of the droplets would contain mRNA (the spike RNA) and so would generate a lot of reads which would take data away from the legitimate cells.

  • How do I schedule a run? - Contact the NGSC lab staff to see when they and/or the 10x controller are available. The NGSC website contains a link to the library prep calendar, see here.

  • What else? - let us know

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