2. Data Preparation

Capfinder requires detailed information about base movements during sequencing, which is captured in the "moves table". This information is crucial for accurate cap type prediction.

Requirements

Raw POD5 files from your sequencing run
Dorado basecaller (version 0.7.0 or later recommended)
Samtools (version 1.18 or later recommended)
A transcriptome reference

Important Note

Dorado Live Basecalling does not generate the necessary moves information. Therefore, post-data acquisition standalone basecalling and alignment process is required to produce the moves information in the alignment SAM/BAM file.

Basecalling Process

To generate the required data, we use a custom script that performs the following steps:

Basecalls the raw POD5 files using Dorado
Emits SAM format output with moves information
Aligns the basecalled reads to a reference genome
Converts the output to a sorted and indexed BAM file

Basecalling Script

Below is a script that automates this process. You'll need to adjust the paths and settings to match your environment:

basecall.sh

#!/bin/bash
# Please edit to reflect your settings
DORADO="/path/to/bin/dorado"
SAMTOOLS="/path/to/samtools"
POD5_DIR="/path/to/pod5_dir"
REF="/path/to/ref.fa"
MODEL_NAME="rna004_130bps_sup@v5.0.0"
MODEL_DIR="/path/to/download/doardo/model"
OUTPUT_DIR="/path/to/save/basecalled/data"
DEVICE="cuda:all" # For Dorado to use GPU

#---------------------- DO NOT EDIT BELOW THIS LINE ---------------------#

# Function to check and download the model if necessary
check_and_download_model() {
    local model_path="$MODEL_DIR/$MODEL_NAME"
    if [ ! -d "$model_path" ]; then
        echo "Model not found. Downloading..."
        "$DORADO" download --directory "$MODEL_DIR" --model "$MODEL_NAME"
    else
        echo "Model already exists. Skipping download."
    fi
}

# Create output directory
mkdir -p "$OUTPUT_DIR"

# Check and download the model if necessary
check_and_download_model

# Run dorado basecaller and pipe directly to samtools for sorting and indexing
echo "Starting basecalling and BAM creation..."
"$DORADO" basecaller "$MODEL_DIR/$MODEL_NAME" "$POD5_DIR/" \
    --recursive \
    --emit-sam \
    --emit-moves \
    --device "$DEVICE" \
    --reference "$REF" | \
"$SAMTOOLS" view -bS - | \
"$SAMTOOLS" sort -o "$OUTPUT_DIR/sorted.bam" -

# Index the sorted BAM file
echo "Indexing the sorted BAM file..."
"$SAMTOOLS" index "$OUTPUT_DIR/sorted.bam"

echo "Basecalling and processing completed. Output files are in $OUTPUT_DIR"
echo "Generated files: sorted.bam and sorted.bam.bai"

To use this script:

Save it to a file (e.g., basecall.sh).
Make it executable:
```
chmod +x basecall.sh
```
Edit the script directly to change any paths or settings as needed.
Run the script:
```
./basecall.sh
```