ATAC-Seq alignment and QC

Alignment workflow and Report page overview

The alignment of each individual sample starts with fastq data, performs QC, aligns reads to the genome using Bowtie2, removes duplicate reads, and creates genomic coverage files. Summary statistics and plots are provided to allow for a quick assessment of data quality and an embedded genome browser provides a direct look at the data.

Results

Under the "Report" tab, a series of interactive and downloadable plots allow for in-depth data exploration

Quality Scores

-Average quality scores at each base of the raw sequencing reads and also after trimming

Number of Reads

-a sankey plot summarizing the trimming and alignment steps

Coverage

-a summary plot of read-coverage throughout the genomeInsert Size

-distribution of the insert-sizes for paired-end data

Gene Body and Transcription Start Site

-Heatmaps and average profiles of normalized signal at TSS's and gene bodies

Genome Browser

-an interactive, embedded IGV browser session displaying the normalized signal bigwig track

*multiple genomic loci can be visualized by specifying them with spaces in-between:

chr8:128,740,266-128,761,729 chr10:3,818,235-3,836,806 chr19:16,433,128-16,438,505

Output Files

Under the "Info" tab, intermediate files produced by the pipeline can be viewed and downloaded:

QC, Trim
trim/<SAMPLE_NAME>.trim.report.html

-a detailed report of raw sequencing quality, base content, estimates of PCR duplication level, insert size distribution, adapter content, and kmer overrepresentationAlign (Bowtie2)
bowtie/<SAMPLE_NAME>.<genome>.bam.bai

-index file of the raw alignment BAM filebowtie/<SAMPLE_NAME>.<genome>.bam

-compressed BAM file of the raw alignmentsRemove chrM Reads
rm_chrM/<SAMPLE_NAME>.<genome>.rm_chrM.bam.bai

-index of the alignment BAM file after mitochonrial removal
rm_chrM/mitochondrial_reads.stats

-text file containing stats on the mitochondrial removalrm_chrM/<SAMPLE_NAME>.<genome>.rm_chrM.bam

-compressed BAM file of the alignments after removing mitochondrial readsRemove duplicates
dedup/<SAMPLE_NAME>.<genome>.rm_chrM.dedup.bam.bai

-index file of the alignments after mitochondrial removal and de-duplication
dedup/duplicate_reads.stats

-text file containing stats on the deduplication
dedup/<SAMPLE_NAME>.<genome>.rm_chrM.dedup.bam

-compressed BAM file of alignments after mitochondrial removal and de-duplication

Insert size
insert_size/<SAMPLE_NAME>.<genome>.rm_chrM.dedup.insert-size-histogram.png

-raw picard tools insert size distribution plot

Coverage
coverage/<SAMPLE_NAME>.<genome>.rm_chrM.dedup.coverage-summary.xls

-summary statistics of genome-wide coverageFilter reads
filter/<SAMPLE_NAME>.<genome>.rm_chrM.dedup.filter-100.bam.bai

-index file of alignments with insert sizes less than 100bp ("filter")
filter/<SAMPLE_NAME>.<genome>.rm_chrM.dedup.filter-100.bam

-compressed BAM file of alignments with insert sizes less than 100bp ("filter")

*insert sizes <100bp correspond to subnucleosomal fragments typically coming from nucleosome-free regions (NFRs)
BigWig
bam_to_bigwig/<SAMPLE_NAME>.<genome>.rm_chrM.dedup.bigwig

-normalized, compressed signal track in bigwig formatBigWig
bam_to_bigwig_filtered/<SAMPLE_NAME>.<genome>.rm_chrM.dedup.filter-100.bigwig-normalized, compressed signal track of subnucleosomal fragments (insert size <100bp) in bigwig format