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Effect of unequal coverage and lack of biological replicates on global 6mA comparisons

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5/5
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Più di una settimana
Idoneità per principianti
25/100
Tipo di issue
Documentazione
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Da chiarire
Stato di attività
Tranquilla
Ambito
bioinformatics

Direzione di ricerca

The issue contains coverage summaries and a one-versus-one 6mA comparison, but names no files, tests, or entry points. Start by reviewing the reported calculation and the relevant modkit documentation; done means providing clear methodological guidance on coverage normalization, subsampling, and the limits of comparisons without biological replicates.

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Descrizione

Hello Modkit developers,

I would like to ask for guidance on comparing global 6mA levels between two Nanopore samples with unequal sequencing coverage and no biological replicates.

Both samples were processed using the same Dorado model, Q15 filtering, reference genome and Modkit settings. A fixed adenine probability threshold of A:0.9394531 was used for both samples to avoid differences caused by dynamic threshold estimation.

I calculated the global 6mA percentage as:

100 × sum(Nmod) / sum(Nvalid)

where:

Nvalid = Nmod + Ncanonical

The results were:

  • barcode04, complete dataset

    • Mean genomic coverage: 203.425×
    • Mean valid coverage per adenine site: 91.639
    • Adenine sites with Nvalid >10: 17,117,572
    • Global 6mA: 0.99147%

  • barcode04, subsampled to approximately 52×

    • Mean valid coverage per adenine site: 24.584
    • Adenine sites with Nvalid >10: 16,683,017
    • Global 6mA: 0.99106%

  • barcode07

    • Mean genomic coverage: 51.723×
    • Mean valid coverage per adenine site: 23.665
    • Adenine sites with Nvalid >10: 16,696,954
    • Global 6mA: 0.94674%

Subsampling barcode04 to approximately the coverage of barcode07 substantially reduced its mean valid coverage per adenine site, as expected. However, it retained approximately 97.46% of the sites that originally had Nvalid >10, and its global 6mA estimate changed only from 0.99147% to 0.99106%.

My questions are:

  1. Is sum(Nmod) / sum(Nvalid) an appropriate genome-wide comparison when samples have substantially different coverage? Does the stability observed after subsampling indicate that coverage is not materially biasing the global 6mA estimate?

  2. For this global comparison, would you recommend using all available reads or subsampling the higher-coverage sample to match the lower-coverage sample?

  3. How reliable is a one-versus-one comparison without biological replicates? Should it only be interpreted as a descriptive comparison between these two samples, and are biological replicates required for robust single-site or DMR inference?

Thank you very much for your help.

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Rust
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