Problems from Trimmomatic trimming

[jt...@tongji.edu.cn]

Hi,

Before using Trinity to analyze my RNA sequences, I use Trimmomatic to remove adapter sequences and bad quality parts.

My RNA sequences are paired end. I run Trimmomatic twice to remove the adapters, one with TruSeq3-PE-2.fa, one with NexteraPE-PE.fa, their adapters sequences are as follows:

TruSeq3-PE-2.fa

>PrefixPE/1

TACACTCTTTCCCTACACGACGCTCTTCCGATCT

>PrefixPE/2

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT

>PE1

TACACTCTTTCCCTACACGACGCTCTTCCGATCT

>PE1_rc

AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTA

>PE2

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT

>PE2_rc

AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC

AGATCGGAAGAGCACACGTCAGAACTCCAGTCACAAGAGGATCTCGTATG

>TruSeq1

AGATCGGAAGAGCACACGTCAGAACTCCAGTCACACCTCAATCTCGTATG

>TruSeq2

AGATCGGAAGAGCACACGTCAGAACTCCAGTCACAGCATGATCTCGTATG

For  NexteraPE-PE.fa,

>PrefixNX/1

AGATGTGTATAAGAGACAG

AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCC 

>PrefixNX/2

AGATGTGTATAAGAGACAG

AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCC 

>Trans1

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG

>Trans1_rc

CTGTCTCTTATACACATCTGACGCTGCCGACGA

>Trans2

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG

>Trans2_rc

CTGTCTCTTATACACATCTCCGAGCCCACGAGAC

Commands are (take NexteraPE-PE.fa as the example):

java -jar /Users/vedgcomb/Desktop/Trimmomatic-0.32/trimmomatic-0.32.jar PE 81R_S1_R1_paired.fastq 81R_S1_R2_paired.fastq 
81R_R1_paired.fastq 81R_R1_unpaired.fastq 81R_R2_paired.fastq 81R_R2_unpaired.fastq ILLUMINACLIP:/Users/vedgcomb/Desktop/Trimmomatic- 
0.32/adapter/NexteraPE-PE.fa:2:30:10 LEADING:20 TRAILING:20 SLIDINGWINDOW:04:24 MINLEN:50

We can get the information reminding us it was successfully finished.

When I use the Fastqc to estimate the quality of sequences obtained after Trimmomatic-treating, it indicates that 'Adapter content ' failed (red cross) and still has much Nextera Transposase Sequences (see attachement).

Anybody can help me to analyze the reason and tell me how to do to remove the adapter sequences.

Thanks a million

Jiangtao