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Old 11-14-2016, 02:14 PM   #1
dimo
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Default WXS kmer over-representation post trimming.

Greetings and salutations,
I am attempting to analyse some human Illumina WXS data (SureSelect) and am getting some unfamiliar kmer over-representation. Usually post trimming (trimmomatic) I get quite good removal of kmers but with this latest data I can't seem to get rid of the 3-primer kmers. My question is in two parts.

1) Should I worry about this or just continue alignment/variant calling.

2) If I should worry about, how can I trim these successfully?

My trimmomatic parameters and pre/post trimming images are below, but I am probably missing something super obvious.

Thanks in advance.



trimmomatic-0.36.jar -phred33 1_ATGCCTAA_L001_R1.fastq.gz 1_ATGCCTAA_L001_R2.fastq.gz output_forward_paired.fq.gz output_forward_unpaired.fq.gz output_reverse_paired.fq.gz output_reverse_unpaired.fq.gz ILLUMINACLIP:adapters/TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:75




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Old 11-15-2016, 12:51 PM   #2
Brian Bushnell
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Maybe you are using the wrong adapter sequences. With the BBMap package, you can discover adapter sequence like this:

Code:
bbmerge.sh in1=1_ATGCCTAA_L001_R1.fastq.gz in2=1_ATGCCTAA_L001_R2.fastq.gz outa=adapters.fa reads=8m
Alternately, you can use the set of adapter sequences included with the package in bbmap/resources/adapters.fa, which is pretty complete. I recommend trimming with BBDuk which is more sensitive than other trimming tools, in my tests:

Code:
bbduk.sh in1=1_ATGCCTAA_L001_R1.fastq.gz in2=1_ATGCCTAA_L001_R2.fastq.gz out1=trimmed1.fq.gz out2=trimmed2.fq.gz ref=adapters.fa ktrim=r k=23 mink=11 hdist=1 tbo tpe minlen=75
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Old 11-15-2016, 03:07 PM   #3
dimo
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Appreciate the response Brian, will run through BBMap and report back.
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Old 11-16-2016, 02:08 PM   #4
dimo
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Quote:
Originally Posted by Brian Bushnell View Post
Maybe you are using the wrong adapter sequences. With the BBMap package, you can discover adapter sequence like this:

Code:
bbmerge.sh in1=1_ATGCCTAA_L001_R1.fastq.gz in2=1_ATGCCTAA_L001_R2.fastq.gz outa=adapters.fa reads=8m
Alternately, you can use the set of adapter sequences included with the package in bbmap/resources/adapters.fa, which is pretty complete. I recommend trimming with BBDuk which is more sensitive than other trimming tools, in my tests:

Code:
bbduk.sh in1=1_ATGCCTAA_L001_R1.fastq.gz in2=1_ATGCCTAA_L001_R2.fastq.gz out1=trimmed1.fq.gz out2=trimmed2.fq.gz ref=adapters.fa ktrim=r k=23 mink=11 hdist=1 tbo tpe minlen=75

Worked an absolute treat. You are a gentleman and a scholar, thanking you.
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