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Application of single cell sequencing in immune cell analysis

A single-cell sequencing and immune cell technology, applied in the application field of single-cell sequencing in immune cell analysis

Pending Publication Date: 2020-02-21
SUZHOU SINGLERON BIOTECHNOLOGIES LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Selecting different specific markers to identify trace amounts of ctDNA in blood samples based on different starting amounts of cell-free DNA (cfDNA) is a current technical challenge

Method used

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  • Application of single cell sequencing in immune cell analysis
  • Application of single cell sequencing in immune cell analysis
  • Application of single cell sequencing in immune cell analysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Construction and sequencing of single-cell transcriptome

[0047] (1) Single cell pretreatment

[0048] use Massive Single Cell mRNA Sequencing Preprocessing Kit processes single cells, including the following steps:

[0049] ①Preparation steps:

[0050] Chip preparation: the small hole end of the chip is the sample inlet, and the big hole end is the sample outlet, so that both the big and small holes are filled with 0.02% PBS-Tween liquid, put the chip in a vacuum desiccator for 15 minutes, take it out and put it at room temperature Let it stand for 15 minutes to ensure that there are no air bubbles in the chip;

[0051] Lysis buffer (lysis buffer) and magnetic beads (beads) preparation: prepare the lysate on ice, and wash the magnetic beads with PBS buffer;

[0052] Cell preparation: resuspend the cells in PBS, and adjust the corresponding cell concentration according to the expected number of cells;

[0053] ② Chip operation:

[0054] Inject the resu...

Embodiment 2

[0063] Embodiment 2 biological information analysis

[0064] The sequencing data analysis method of this embodiment includes:

[0065] (1) Barcode quality control analysis

[0066] Before the overall quality control, first perform barcode data quality analysis, filter out the cellbarcode with low sequencing quality to ensure the accuracy of subsequent analysis, filter the number of bases in the barcode with a quality value lower than 14 or greater than 1; in addition, for UMI The subsequent reads without polyA are also filtered;

[0067] In order to obtain high-quality data for subsequent analysis, use fastp to trim Reads2 and filter reads. The filtering content is as follows:

[0068] Truncating the end adapter sequence;

[0069] Remove more than 5 reads containing N (N means that the base information cannot be determined);

[0070] According to the 4-base sliding window from the 5' end to the 3' end of the reads, the 3' end sequence of the window whose average quality va...

Embodiment 3

[0080] Example 3 Cell Subtype Annotation

[0081] Using the semi-automated annotation of cell subgroups independently developed by Xinge, as well as the reference data set and knowledge base in the Xinge database to annotate cell subtypes, the steps are as follows:

[0082] (1) Semi-automatic cell subgroup annotation: According to the average expression level and cell expression percentage of the marker gene of immune cells in each cluster, the cell type of each cluster is obtained, and this work is completed by running a script;

[0083] (2) Manual verification:

[0084] First, understand the sample type (species, tissue / peripheral blood, tumor / adjacent cancer) based on the sample information, and then judge the main cell type of immune cells based on the clinicopathological information related to the sample (stage, curative effect, etc.);

[0085] According to the gene expression in the differentially expressed gene list of each subgroup, evaluate the possible subtypes. The...

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Abstract

The invention provides an application of single cell sequencing in immune cell analysis. The immune cells comprise any one or a combination of at least two of T cells, mononuclear cells, natural killer cells, B cells, macrophages, granulocytes, mast cells, megakaryocytes or dendritic cells. Wherein the immune cell analysis comprises analysis of any one or a combination of at least two of types, states or proportions of immune cells and / or immune cell subtypes. In the present invention, single cell sequencing is applied to the immune cell analysis, a method comprises the following steps: firstly, obtaining a sequencing result of a sample through single cell sequencing, then obtaining a distribution state, subtype composition and a cell track of immune cells by adopting a biological information analysis method, annotating the cell subtypes, and realizing analysis on the types, states and proportions of the immune cells and / or the immune cell subtypes by comparing different results.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to the application of single cell sequencing in immune cell analysis. Background technique [0002] It is difficult to perform biopsy on primary tumors. As one of the most widely used biopsy methods, puncture cannot guarantee sufficient materials every time in clinical application, and there are also high requirements for operating doctors and possible It has limitations such as artificially causing tumor cell metastasis, causing pain to patients, and easily causing complications. Using blood as a clinical sample has significant advantages: blood is easy to obtain and has little harm to patients, and some components in blood reflect different pathological states. Therefore, analyzing blood samples can help diagnose diseases in patients. [0003] Liquid biopsy analysis can be applied to the detection and characterization of minimal residual disease (MRD), real-time monitoring of cancer de...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869C12Q1/6886C12M1/34G16B25/10G16B30/00G16B50/30
CPCC12Q1/6869C12Q1/6886G16B25/10G16B30/00G16B50/30C12Q2600/158C12Q2600/136
Inventor 范珏陈云美方南高月
Owner SUZHOU SINGLERON BIOTECHNOLOGIES LTD
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