RNA-seq bam file normalization
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5 weeks ago
maplewj ▴ 10

I'm a beginner in bioinformatics and I have a question. I've performed pre-processing for RNA-seq analysis, used hisat2 for read alignment, and created sorted.bam and sorted.bam.bai files with samtools. I want to visualize this using IGV. Here's where I'm curious: I have 3 replicates for each condition. When visualizing with IGV, is it okay to show just one replicate, or do I need to merge the bam files for replicates of the same condition and show the combined bam file? Another question is about the different number of reads per sample. Is it okay to compare bam files directly in IGV without a normalization process, or is there a normalization process included during the hisat2 step? I would really appreciate an answer.

RNA-seq bam normalization • 294 views
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Why would you even visualize RNA-seq on the IGV, just curious? What is the actual analysis goal? You're not going to find differences by looking at the IGV anyway, most likely.

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In the case of RNA-seq, isn't it common to visualize the BAM file in IGV to check? I applied group autoscale for comparison and used IGV to ensure that the read mapping proceeded without issues. For example, just as ATAC-seq is compared using coverage BAM files and bigwig, isn't this process suitable for RNA-seq as well?

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I visualize RNA-seq BAMs in IGV to look at coverage (i.e. maybe only the 5' end of a gene is covered?), PCR duplicates, etc.

In any case, if they're replicates, just combine them (and there's no need to normalize since you're looking at individual reads and how they stack up). You're not going to be doing any quantitative analysis on IGV most likely.

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