single cell 基础笔记（一）

课程地址 https://hemberg-lab.github.io/scRNA.seq.course/index.html

2 Introduction to single-cell RNA-seq

2.5 Challenges
sequencing libraries 即 different cells

The main sources of discrepancy between the libraries are:
Amplification (up to 1 million fold)
Gene ‘dropouts’ in which a gene is observed at a moderate expression level
in one cell but is not detected in another cell (Kharchenko, Silberstein, and Scadden 2014).

libraries之间的差别主要来自于：RNA规模（扩增效率）的不同及gene dropouts（基因子啊有些cell中表达值缺失）

In both cases the discrepancies are introduced due to low starting
amounts of transcripts since the RNA comes from one cell only.
Improving the transcript capture efficiency and reducing the
amplification bias are currently active areas of research. However, as
we shall see in this course, it is possible to alleviate some of these
issues through proper normalization and corrections.

差异是由于一个cell转录起始量较低。
提高转录捕获效率（transcript capture efficiency）及减少放大偏差（amplification bias）
通过适当的标准化及纠正，也可以缓解这些问题。

2.7 What platform to use for my experiment?

For example, if one is interested in characterizing the composition of
a tissue, then a droplet-based method which will allow a very large
number of cells to be captured is likely to be the most appropriate.
On the other hand, if one is interesting in characterizing a rare
cell-population for which there is a known surface marker, then it is
probably best to enrich using FACS and then sequence a smaller number
of cells.

单细胞测序的对照 https://www.sohu.com/a/122201336_390793

为了更好估计和消除单细胞测序文库间的技术（系统）误差，现有两种定量标准被广泛采用，即spike-ins和UMIs。使用这两种对照是为了辅助规范（normalization）不同细胞间的基因表达水平。

Spike-ins
Spike-ins是已知浓度的外源RNA分子。在单细胞裂解液中加入Spke-ins后，再进行反转录。最广泛使用的Spike-ins是External
RNA Control Consortium (ERCC)提供的合成spikes。其包含96个不同长度和GC含量的mRNA分子 (Jiang et al. 2011)。
但是spike-ins的使用浓度通常很高，结果会占据很大比例的测序reads。最新的Drop-seq技术也还没不能加入spike-ins。
UMIs
另一种标准化方法是使用 Unique Molecular Identifiers (UMIs)(Kivioja et al. 2012).
UMIs是一种随机条形码（barcode）序列，长度在4-20 bp之间。
在扩增步骤之前（通常在反转录期间