Spike-in normalization

CHAMPAGNE supports ChIP-seq experiments that spike-in reads from alternate species. The data are normalized based on the number of reads aligning to the spike-in genome to account for differences in sequencing depth or other technical variations between samples.

Samples are grouped by antibody when calculating the scaling factors. Inputs are excluded from this calculation.

Spike-in options

You must set the spike_genome parameter to the name of a supported genome (e.g. dmelr6.32, ecoli_k12) that is different from the genome used for the main analysis. When using spike-in normalizations, we recommend setting deeptools_normalize_samples to None so that additional normalization isn’t performed (see this discussion). You can separately set the normalization method for input samples with the deeptools_normalize_input parameter.

View the spike-in options for a full list of parameters that can be set for spike-in normalization.

normalization method

CHAMPAGNE implements two methods for spike-in normalization:

• delorenzi: This method uses deepTools multiBamSummary --scalingFactors to calculate the scaling factors, which is similar to the method described here: 10.1101/gr.168260.113. To use this method, set the spike_norm_method parameter to delorenzi (default).

• guenther: This method is described in the supplementary material of 10.1016/j.celrep.2014.10.018. Reads are scaled by the minimum number of reads aligning to the spike-in genome across all samples. To use this method, set the spike_norm_method parameter to guenther.

Examples

Using the delorenzi normalization method with E. coli as the spike-in genome:

champagne run \
    --output /data/$USER/champagne_project/ \
    --genome hg38 \
    --input assets/samplesheet_full_spikein.csv \
    --spike_genome ecoli_k12 \
    --deeptools_normalize_samples None \
    --spike_norm_method delorenzi

Using the guenther normalization method with D. melanogaster as the spike-in genome:

champagne run \
    --output /data/$USER/champagne_project/ \
    --genome hg38 \
    --input assets/samplesheet_full_spikein.csv \
    --spike_genome dmelr6.32 \
    --deeptools_normalize_samples None \
    --spike_norm_method guenther