I am analysing Nextera paired-end sequencing data and have discovered that ~25% of the reads align in an RF orientation, but I'm not sure why? According to the biochemistry of the sample preparation there should only be FR orientated reads?
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There are tools out there that will incorrectly call read orientation if the insert size is shorter than read length. How, specifically, did you do preprocessing (such as adapter-trimming), mapping, and calculate the orientations?
Also, is this a Nextera fragment or LMP library? The LMP libraries can produce output in either orientation.Last edited by Brian Bushnell; 02-27-2015, 04:18 PM.
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I trimmed the adapters using Trimmomatic in PE mode using the Nextera-PE adapters provided. I then mapped the trimmed reads using Bowtie2, with max insert size set to 2000. I then removed PCR duplicates using Picard's MarkDuplicates. I then wanted to look at the insert distribution size and that's when Picard's CollectInsertSizeMetrics reported that I had both RF and FR orientated reads. If I plot a graph of the RF insert sizes, most of them are the size of my reads (~125) but I do still get a lot which are significanly bigger (~500-900bp). It's a bit odd that 75% map in the correct FR orientation, and 25% map in the RF orientation. At first I thought this may be to do with the tagmentation process used by the transposomes. The transposomes are dimers made up of two monomers, each of which has a sinle primer attached. The monomers are made by mixing them in a solution with 50% forward primers and 50% reverse primers. The monomers then dimerise and are used for tagmentation. That means that the 25% of the transposome dimers have two forward primers, 25% have two reverse and 50% have both the forward and reverse. The sample is from an ATAC-seq experiment, it does not have a circularised fragment step, so I don't think it is a LMP library, but a Nextera fragment
Commands used:
# Trimming
java -jar Trimmoamtic.jar PE \
-threads 1 \
sample_1.fastq \
sample_2.fastq \
sample_1P.fastq \
sample_1U.fastq \
sample_2P.fastq \
sample_2U.fastq \
ILLUMINACLIP:NexteraPE-PE.fa:2:30:10:1:true \
LEADING:20 \
TRAILING:20 \
SLIDINGWINDOW:4:15 \
MINLEN:15
# Mapping
bowtie2 -X 2000 \
-p 4 \
-x genome \
-1 sample_1P.fastq \
-2 sample_2P.fast \
-S sample.sam
# Remove PCR duplicates
java -Xmx2g -XX:ParallelGCThreads=4 -jar Picard.jar MarkDuplicates \
I=sample.sorted.bam \
O=sample.sorted.rmdup.bam \
M=sample.sorted.rmdup.pcrMetrics \
REMOVE_DUPLICATES=true \
ASSUME_SORTED=true \
VALIDATION_STRINGENCY=LENIENT
# Collect insert sizes
java -jar Picard.jar CollectInsertSizeMetrics \
I=sample.sorted.rmdup.bam \
O=sample_insertsize.metrics \
H=sample_insertsize.pdf \
VALIDATION_STRINGENCY=LENIENT
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wrong RF orientated reads in atac-seq bam file
Originally posted by jmacrm91 View PostI am analysing Nextera paired-end sequencing data and have discovered that ~25% of the reads align in an RF orientation, but I'm not sure why? According to the biochemistry of the sample preparation there should only be FR orientated reads?
Karen
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