minimap2 2.27 not outputting MD tag

[ddubocan]

Hi,

Recently upgraded to 2.27 via bioconda and noticed that even when including the --MD tag , no MD tag was output.

Downgrading back down to 2.26 fixed the issue, and MD tags were output again.

[lh3]

This is caused by the same bug #1181. I will cut a new release later this week.

[wdg118]

Hi @lh3 ,

Was this ever fixed ? I tried @ddubocan 's solution but I still don't get the MD tags.

I ran:

#!/bin/bash

# Define paths
REFERENCE="/home/jupyter/GRCh38.primary_assembly.genome.fa"
FASTQ_DIR="/home/jupyter/samples"
OUTPUT_DIR="/home/jupyter/talon_output/sam_files"

# Create output directory if it doesn't exist
mkdir -p "$OUTPUT_DIR"

# Loop through FASTQ files and align each to the reference genome
for FASTQ_FILE in "$FASTQ_DIR"/pychopper_BC*Merged.fastq; do
    # Extract the base name for the output file
    BASE_NAME=$(basename "$FASTQ_FILE" .fastq)
    
    # Define output SAM file path
    OUTPUT_SAM="$OUTPUT_DIR/${BASE_NAME}.sam"
    
    # Run minimap2 to align reads and generate SAM file with MD tags
    minimap2 -ax map-ont --MD -t 8 "$REFERENCE" "$FASTQ_FILE" > "$OUTPUT_SAM"
    
    echo "Alignment completed for $FASTQ_FILE."
done

echo "All alignments completed."

and then when running:

#!/bin/bash

for sam_file in /home/jupyter/talon_output/sam_files/*.sam; do
    echo "Checking $sam_file for MD tags..."
    missing_tags=$(grep -v "^@" $sam_file | grep -v "MD:Z" | wc -l)
    
    if [ "$missing_tags" -eq "0" ]; then
        echo "All reads in $sam_file have MD tags."
    else
        echo "$missing_tags reads in $sam_file are missing MD tags."
    fi
done

I get:

Checking /home/jupyter/talon_output/sam_files/pychopper_BC01Merged.sam for MD tags...
11456 reads in /home/jupyter/talon_output/sam_files/pychopper_BC01Merged.sam are missing MD tags.
Checking /home/jupyter/talon_output/sam_files/pychopper_BC02Merged.sam for MD tags...
19734 reads in /home/jupyter/talon_output/sam_files/pychopper_BC02Merged.sam are missing MD tags.
Checking /home/jupyter/talon_output/sam_files/pychopper_BC03Merged.sam for MD tags...
16840 reads in /home/jupyter/talon_output/sam_files/pychopper_BC03Merged.sam are missing MD tags.
Checking /home/jupyter/talon_output/sam_files/pychopper_BC04Merged.sam for MD tags...
17670 reads in /home/jupyter/talon_output/sam_files/pychopper_BC04Merged.sam are missing MD tags.
Checking /home/jupyter/talon_output/sam_files/pychopper_BC05Merged.sam for MD tags...
9549 reads in /home/jupyter/talon_output/sam_files/pychopper_BC05Merged.sam are missing MD tags.
Checking /home/jupyter/talon_output/sam_files/pychopper_BC06Merged.sam for MD tags...
12603 reads in /home/jupyter/talon_output/sam_files/pychopper_BC06Merged.sam are missing MD tags.
Checking /home/jupyter/talon_output/sam_files/pychopper_BC07Merged.sam for MD tags...
4052 reads in /home/jupyter/talon_output/sam_files/pychopper_BC07Merged.sam are missing MD tags.
Checking /home/jupyter/talon_output/sam_files/pychopper_BC08Merged.sam for MD tags...
3989 reads in /home/jupyter/talon_output/sam_files/pychopper_BC08Merged.sam are missing MD tags.
Checking /home/jupyter/talon_output/sam_files/pychopper_BC09Merged.sam for MD tags...
6884 reads in /home/jupyter/talon_output/sam_files/pychopper_BC09Merged.sam are missing MD tags.
Checking /home/jupyter/talon_output/sam_files/pychopper_BC10Merged.sam for MD tags...
7445 reads in /home/jupyter/talon_output/sam_files/pychopper_BC10Merged.sam are missing MD tags.
Checking /home/jupyter/talon_output/sam_files/pychopper_BC11Merged.sam for MD tags...
5802 reads in /home/jupyter/talon_output/sam_files/pychopper_BC11Merged.sam are missing MD tags.
Checking /home/jupyter/talon_output/sam_files/pychopper_BC12Merged.sam for MD tags...
2064 reads in /home/jupyter/talon_output/sam_files/pychopper_BC12Merged.sam are missing MD tags.
Checking /home/jupyter/talon_output/sam_files/pychopper_BC13Merged.sam for MD tags...
3195 reads in /home/jupyter/talon_output/sam_files/pychopper_BC13Merged.sam are missing MD tags.

I'm confused as to what is going on here. I need these MD tags to run TALON.

[lh3]

Yes, fixed and released.

[wdg118]

Odd that I'm getting a lot of reads without MD tags. I don't know what's going on here as I ran Minimap2 with --MD.