Add considerations and controls lecture notes

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@@ -39,3 +39,31 @@ Metagenomics still faces some challenges, mostly due to the sequencing process:
 - Quantification of the pathogens is difficult due to the different genome sizes
 - Assembly of reads from different organisms (chimera formation)
 - Unknown function of novel proteins
+*** Consideration for metagenomic/microbiome projects
+
+A surveillance/microbiome study contains multiple steps. A project starts with a goal/hypothesis, so it is important to think about the whole structure.
+
+**** Timeline
+***** Project design
+- Project approval: we need to contact the relevant authorities
+- Subject recruitment: find out if we need controls
+***** Sampling
+- Sample collection: aseptic collection of samples and inclusion of replicates. We can use empty swabs and tubes as controls, to check for background DNA.
+- Sample handling and storage: freeze the sample immediately, consider making a licquate if the sample is going to be used for multiple examinations.
+***** Laboratory steps
+The different steps would be conducted in different areas, ideally in different rooms, to avoid cross contamination.
+
+- DNA/RNA isolation: clean environment to avoid contamination of the sample. We include blank control tubes, tubes without a sample but processed in the same way. We can also include samples with Spike organisms or Mock communities.
+- Library preparation: we prefer PCR-free approaches, we consider splitting the sample and using 2 different barcodes. We should ideally see the same composition in both samples.
+- Sequencing: the technology we choose depends on the research question (size of genome, number of samples, depth). We might use multiple rounds of sequencing, even considering a pilot study to get an idea of the complexity of our sample.
+***** Analysis
+- DNA sequence analysis: we can map the reads to reference databases (read classification) or use assembly.
+- Statistical analysis: the methods vary according to the research question. We can analyze the diversity, compare the samples using multivariate analysis, compare them to metadata using regression.
+**** Additional considerations
+- Process all the samples in the same way: the processing can introduce changes that make comparing them difficult. Usually the microbial population is not modified, but the abundance of each species can be altered.
+- Randomization of the samples accross sequencing runs: to mix treatment and control groups.
+- Contaminations: they can happen at any level:
+  1. Host: sometimes they might be a majoritary part of the genomes
+  2. Reference genomes
+  3. Environmental background: during sample handling/DNA sequencing
+- Accessibility: upload the sequences and the studies to open-access journals, with all the supplementary information (code, notebooks), to facilitate reproducibility.