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fix headings pre-processing tutorial
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nomadscientist committed Oct 10, 2023
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#### Why do Single Cell sequencing?
## Why do Single Cell sequencing?


Single-cell RNA (scRNA) sequencing is the technological successor to classical "bulk" RNA-seq, where samples are no longer defined at the tissue level but at the individual cell level. The bulk RNA-seq methods seen in [previous hands-on material]({% link topics/transcriptomics/tutorials/ref-based/tutorial.md %}) would give the average expression of genes in a sample, whilst overlooking the distinct expression profiles given by the cell sub-populations due to their heterogeneity.
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# Single-Batch Processing

### Data upload and organization
## Data upload and organization

In this tutorial we will be analysing scRNA-seq data of bone marrow cells taken from a single C57 mouse by *Herman et al.* ({% cite Herman2018 %}) and producing a count matrix that we can use for later analysis.

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#### Confirming Reads in the BAM file
### Confirming Reads in the BAM file

We now have a BAM file of our aligned reads, with cell and UMI barcodes embedded in the read headers. We also have the chromosome and base-pair positions of where these reads are aligned. The can be confirmed by peeking into the BAM file:

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* `nM`: The number of base mismatches in the alignment of this read to the reference (here `1`).


#### Filtering the BAM File
### Filtering the BAM File

If we perform counting on the current BAM file we will be counting all reads, even the undesirable ones such as those that did not align so optimally.

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The first step merely requires us to run the same workflow on each of our batches, using the exact same inputs except for the FASTQ paired data. The second step requires a minimal level of interaction from us; namely using a merge tool and selecting our matrices.


### Data upload and organisation
## Data upload and organisation

The count matrix we have generated in the previous section is too sparse to perform any reasonable analysis upon, and constitutes data only of a single batch. Here we will use more populated count matrices from multiple batches, under the assumption that we now know how to generate each individual one of them using the steps provided in the previous section. This data is available at [`Zenodo`](https://zenodo.org/record/3253142).

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The plot that follows tells us everything we need to know about each of our batches. Each batch is essentially tested against the full set of barcodes in order to assert that only the desired or 'Real' barcodes have been sequenced.

#### Cross-contamination Plot
### Cross-contamination Plot

![Contamination Plots]({% link topics/single-cell/images/scrna-pre-processing/scrna_crosscontamination.png %} "The Pre-filter and Post-filter plots")

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