> For the complete documentation index, see [llms.txt](https://jodyphelan.gitbook.io/tutorials/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://jodyphelan.gitbook.io/tutorials/ngs/sample-qc.md).

# Sample QC

When analysing NGS data for a sample it is a good idea to check the quality of your data. The general pipeline goes FastQ -> BAM -> VCF. At each stage there are a few things to look out for.

## Raw data (fastq)

| Metric        | Explanation                                                                                                                                                                                                                                                                      | Comments                                                                                                                                |
| ------------- | -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | --------------------------------------------------------------------------------------------------------------------------------------- |
| Base quality  | The machine outputs an estimated quality per base that is sequenced. This estimated by the sequencing machine itself (see [here](https://emea.illumina.com/content/dam/illumina-marketing/documents/products/technotes/technote_understanding_quality_scores.pdf) for more info) | Ideally, you want more of the bases to be of high quality (>Q30). Low-quality bases may be incorrect and lead wrong conclusions.        |
| Contamination | Tools like [kraken2](https://ccb.jhu.edu/software/kraken2/) and [centrifuge](https://ccb.jhu.edu/software/centrifuge/manual.shtml) can assign each read to a species or other taxonomic classification. This helps to look for contamination                                     | The more reads assigned to your organism of interest, the better. Drop samples with high levels of contamination if  you can afford to. |

## Alignment (bam)

| Metric             | Explanation                                                                                                                                                   | Comments                                                                                                                                                                                                                                                                                                                                                                                                                          |
| ------------------ | ------------------------------------------------------------------------------------------------------------------------------------------------------------- | --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- |
| % reads mapping    | We can check the proportion of the total reads which align to the reference. This will tell you if what you sequenced is actually what you wanted to sequence | In a best-case scenario, all your reads should align to the reference. Lower numbers may indicate contamination or a reference which is divergent from your strain.                                                                                                                                                                                                                                                               |
| Reference coverage | Tools like samtools and bedtools can estimate how much of the reference is covered by our data.                                                               | This also looks at the bam file but looks at it from the perspective of the reference. We want as much of the reference covered by at least 5 reads so that we can confidently call variants and look for differences. Depending on your question you might want all of the genome covered, or a selection of genes. If the value is low, this does not mean that your data is bad quality, but that you don't have enough of it. |