Hi, I'm totally new here and totally new to bioinformatic (I think I technically started learning this last week)
Story goes like this, I got my atac-seq fastq data last Monday, and I started to turn these fastq files into peaks. I learnt how to trim, align and visualize my data and a little bit QC afterward.
I chose Trimmomatic in the galaxy of my university (Illuminaclip Nextera pair end adapter) to trim my fastq file, and got 4 files, 2 paireds, and 2 unpaireds. I only aligned my paired fastq files with Bowtie2 -X 2000, and got mapped rate as 90%. I converted the BAM files (default output of the bowtie2 in our galaxy) into bedgraph then tdf in IGV for visualization. I plotted the distribution of the reads surrounding the TSS of my annotated genome and got highly enrichment near TSS.
OK, weird thing happened. I plotted the insert distribution using picard tool, and got this plot:
It appeared that I lost all the inserts smaller than 120 bp which is actually the nucleosome-free-regions that I need most.
Then I guessed I must have some data that were not mapped, so I went back to my fastq file, and found those unpaired data generated from Trimmomatic are huge. For example, each of the paired file is 8 GB, and one unpaired R1 file is 4.5GB, the other is 10mb.
I wonder whether my nucleosome-free-regions just lied in these unpaired data, and how I can combine this unpaired data with my paired data generated from Trimmomatic?
Thanks,
Huan
Map unpaired reads as single-end data. You should have a look at what sequences get trimmed off – low quality, 3'-end adapter or something else? To investigate the issue, an alternative is to map untrimmed reads with a local mapper such as bowtie2 --local or bwa-mem. These mappers won't map adapter sequences. Sometimes this may be easier when you are not sure what trimmomatic is doing to your data.
Sorry, not sure how to upload the figure. You may find figure through this "http://postimg.org/image/m1fmud4u9/"
Well congratulations on your first week, looks like you already learned a lot. Good luck!