Hello All,
This might sound a very basic question to some of you. I am analyzing MiSeq deep sequencing targeted amplicons (human genome, paired ended). I used the MiSeq Reporter TruSeq Amplicon workflow and I got my output files (bam and vcf). Now I would like to process the data using bwa mem + samtools. I was wondering if primer trimming is performed during the fastq generation step by MiSeq Reporter. I am worried about the presence of PCR primers in the sequenced fragment. The presence of them could skew the variant allele frequency. So, I would like to use ULSO and DLSO sequences present in the manifest file in trimmomatic and trim them. What do you think?
I'll really appreciate any help you could can give me,
Luciano
By default MiSeq workflow performs adapter trimming during FASTQ file production. Manual primer trimming is usually recommended when the read length of the sequencing machine is longer than the molecule that is sequenced (for example miRNA). However, if you have concerns about residual primers sequences you can use separate tools like FASTX-toolkit or cutadapt. Also GATK has the clipreads function to perform quality-base or sequence-based trimming and the ReadAdaptorTrimmer function. This latter take bam file from paired-end sequencing as input and automatically determines adaptor to be trimmed. Take a look to GATK documentation for further details.
I would have a look using fastqc. This is a tool that looks on a part of your reads and let you know if there is still some adapters and if so which ones. In addition, it provides you with information regarding the quality of your reads.
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