RADCamp Latin America 2026 Part II (Bioinformatics)

Day 2 (AM)

Overview of the activities:

Demultiplex Empirical Data

Lead: Isaac (Could take all morning)

The first step in a RADSeq assembly is to evaluate the quality of the raw data. It is typically more useful to do quality control on a per-sample basis rather than on the full undemultiplexed raw data file, so before we run fastqc we need to run ipyrad2 step 1 to demux raw data to samples.

Make a new directory and a new ipyrad2 params file for your empirical assembly

cd ~
mkdir <your_assembly_name>
cd <your_assembly_name>
ipyrad2-classic -n <your_assembly_name>

Edit params file to point to raw fastq data and barcodes file

Edit your new params file and set raw_fastq_path and barcodes_path to the full path where these files are found.

Run step 1 to demultiplex your data

ipyrad2-classic -p params-<your_assembly_name> -s 1 -c 16

Quick check of demux process

Demux fastq files are stored in the *_fastqs directory, so take a look in this directory to get a quick feel for how the data is distributed among samples:

# `-l` gives a detailed (long) list
# `-h` shows file sizes in human readable format 
ls -lh <your_assmbly_name>_fastqs

Question: Do most of your R1/R2 files for each sample show about the same size? Or are some much bigger or much smaller than average?

TODO FINISH

Empirical Data QC

Lead: Isaac (45’)

Now we will run fastqc on a couple of samples from the empirical data. To do this we will first change directory (cd) to where the demultiplexed samples live, inside ~/empirical/*_fastqs. Consult the fastqc exercise from yesterday for how to run and interpret results.

In the remaining time choose 1-2 samples to run fastqc on both R1 and R2. After the runs finish, inspect the results and try to come to a conclusion about what the results indicate.

Be prepared to answer the following questions:

Coffee break

Briefly report back on fastqc results

Empirical assembly

You already started your empirical assembly when you ran Step 1 to demux your raw data to samples, so now you can continue with steps 2-5 to complete the assembly.