GPTomics/bio-rna-quantification-alignment-free-quant

Version Compatibility

Reference examples tested with: Salmon 1.10+, fastp 0.23+, kallisto 0.50+, pandas 2.2+

Before using code patterns, verify installed versions match. If versions differ:

• CLI: <tool> --version then <tool> --help to confirm flags

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.

Alignment-Free Quantification

"Quantify gene expression without alignment" -> Estimate transcript abundances directly from FASTQ reads using pseudo-alignment or selective alignment, bypassing genome mapping.

• CLI: salmon quant -i index -l A -1 R1.fq.gz -2 R2.fq.gz -o quant/, kallisto quant -i index -o output R1.fq.gz R2.fq.gz

Quantify transcript abundance directly from FASTQ reads using pseudo-alignment (kallisto) or selective alignment (Salmon).

Salmon Workflow

Build Index

# Download transcriptome FASTA
# Ensembl: Homo_sapiens.GRCh38.cdna.all.fa.gz

# Basic index (fast, less accurate)
salmon index -t transcripts.fa -i salmon_index

# Decoy-aware index (recommended for accuracy)
# First, create decoys from genome
grep "^>" genome.fa | cut -d " " -f 1 | sed 's/>//g' > decoys.txt
cat transcripts.fa genome.fa > gentrome.fa
salmon index -t gentrome.fa -d decoys.txt -i salmon_index -p 8

Quantify Samples

# Paired-end reads
salmon quant -i salmon_index -l A \
    -1 sample_R1.fastq.gz -2 sample_R2.fastq.gz \
    -o sample_quant -p 8

# Single-end reads
salmon quant -i salmon_index -l A \
    -r sample.fastq.gz \
    -o sample_quant -p 8

Key flags:

• -l A - Automatically detect library type
• -p - Number of threads
• --validateMappings - More accurate (default in recent versions)
• --gcBias - Correct for GC bias
• --seqBias - Correct for sequence-specific bias

Library Types

| Code | Description | |------|-------------| | A | Automatic detection (recommended) | | ISR | Inward, stranded, read 1 from reverse | | ISF | Inward, stranded, read 1 from forward | | IU | Inward, unstranded |

Batch Processing

for sample in sample1 sample2 sample3; do
    salmon quant -i salmon_index -l A \
        -1 ${sample}_R1.fastq.gz -2 ${sample}_R2.fastq.gz \
        -o ${sample}_quant -p 8
done

Output Files

sample_quant/
├── quant.sf           # Main quantification file
├── aux_info/          # Auxiliary information
├── cmd_info.json      # Command used
├── lib_format_counts.json  # Library format detection
└── logs/              # Log files

quant.sf format:

Name                    Length  EffectiveLength TPM         NumReads
ENST00000456328.2       1657    1477.000        0.000000    0.000
ENST00000450305.2       632     452.000         12.345678   156.789

kallisto Workflow

Build Index

kallisto index -i kallisto_index transcripts.fa

Quantify Samples

# Paired-end
kallisto quant -i kallisto_index -o sample_quant \
    sample_R1.fastq.gz sample_R2.fastq.gz

# Single-end (must specify fragment length)
kallisto quant -i kallisto_index -o sample_quant \
    --single -l 200 -s 20 sample.fastq.gz

# With bootstraps (for sleuth)
kallisto quant -i kallisto_index -o sample_quant -b 100 \
    sample_R1.fastq.gz sample_R2.fastq.gz

Key flags:

• -b - Number of bootstrap samples
• -t - Number of threads
• --single - Single-end mode
• -l - Estimated fragment length (single-end)
• -s - Fragment length standard deviation

Output Files

sample_quant/
├── abundance.tsv      # Main quantification (text)
├── abundance.h5       # HDF5 format (for sleuth)
└── run_info.json      # Run information

abundance.tsv format:

target_id               length  eff_length  est_counts  tpm
ENST00000456328.2       1657    1477.00     0.00        0.000000
ENST00000450305.2       632     452.00      156.79      12.345678

Salmon vs kallisto

| Feature | Salmon | kallisto | |---------|--------|----------| | Speed | Fast | Fastest | | Accuracy | Higher | Good | | GC bias correction | Yes | No | | Decoy sequences | Yes | No | | Memory usage | Moderate | Low |

Recommendation: Use Salmon for production, kallisto for quick exploratory analysis.

Combining Results

# Salmon: use tximport in R
# kallisto: use tximport or sleuth

# Quick Python combination
python << 'EOF'
import pandas as pd
from pathlib import Path

samples = ['sample1', 'sample2', 'sample3']
tpm_data = {}
counts_data = {}

for sample in samples:
    quant_file = Path(f'{sample}_quant/quant.sf')  # Salmon
    # quant_file = Path(f'{sample}_quant/abundance.tsv')  # kallisto
    df = pd.read_csv(quant_file, sep='\t', index_col=0)
    tpm_data[sample] = df['TPM']
    counts_data[sample] = df['NumReads']  # or est_counts for kallisto

tpm_matrix = pd.DataFrame(tpm_data)
counts_matrix = pd.DataFrame(counts_data)
tpm_matrix.to_csv('tpm_matrix.csv')
counts_matrix.to_csv('counts_matrix.csv')
EOF

Quality Checks

# Check mapping rate from Salmon logs
grep "Mapping rate" sample_quant/logs/salmon_quant.log

# Check library type detection
cat sample_quant/lib_format_counts.json

Good metrics:

• Mapping rate > 70%
• Consistent library type across samples

Common Issues

Low mapping rate:

• Wrong transcriptome version
• Contamination in samples
• Wrong library type

Inconsistent library types:

• Mixed library preparations
• Sample swap

Related Skills

• read-qc/fastp-workflow - Upstream preprocessing
• rna-quantification/tximport-workflow - Import results to R
• rna-quantification/count-matrix-qc - QC of quantification
• differential-expression/deseq2-basics - Downstream analysis