MeRIP-seq Preprocessing
Alignment with STAR
Build index (once)
STAR --runMode genomeGenerate
--genomeDir star_index
--genomeFastaFiles genome.fa
--sjdbGTFfile genes.gtf
Align IP and input samples
for sample in IP_rep1 IP_rep2 Input_rep1 Input_rep2; do
STAR --genomeDir star_index
--readFilesIn ${sample}_R1.fastq.gz ${sample}R2.fastq.gz
--readFilesCommand zcat
--outSAMtype BAM SortedByCoordinate
--outFileNamePrefix ${sample}
done
QC Metrics
Index BAMs
for bam in *Aligned.sortedByCoord.out.bam; do samtools index $bam done
Check IP enrichment
Good MeRIP: IP should have peaks, input should be uniform
samtools flagstat IP_rep1_Aligned.sortedByCoord.out.bam
IP/Input Correlation
import deeptools.plotCorrelation as pc
Check replicate correlation
multiBamSummary bins
-b IP_rep1.bam IP_rep2.bam Input_rep1.bam Input_rep2.bam
-o results.npz
plotCorrelation -in results.npz
--corMethod spearman
-o correlation.png
Related Skills
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read-qc/quality-reports - Raw read quality assessment
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read-alignment/star-alignment - General alignment concepts
-
m6a-peak-calling - Next step after preprocessing