Question: Adapter sequence removal from raw reads
0
gravatar for Bioinfonext
3.8 years ago by
Bioinfonext250
Korea
Bioinfonext250 wrote:

I have a pair end raw read data. I want to remove adaptor and low-quality raw reads using Trimmomatic.

Our raw read also has some bar code like this:

sample  Sample description                 barcode
216_5W_Ca1  line 216 5week Cam rep1 AACCAG 
216_5W_Ca2  line 216 5week Cam rep2 GCACTA 
216_5W_Co1  line 216 5week Cortrep1 AGTGAG 
216_5W_Co2  line 216 5week Cortrep2 GTGCTT 
216_5W_Pa1  line 216 5week Paren rep1   TGGTGA 
216_5W_Pa2  line 216 5week Parenrep2    ACCTCA

For trimmomatic I am using below cammnad and the attached adaptor sequence file, but it do not include bar code.

java -jar trimmomatic-0.35.jar PE -phred33 input_forward.fq.gz input_reverse.fq.gz output_forward_paired.fq.gz output_forward_unpaired.fq.gz output_reverse_paired.fq.gz output_reverse_unpaired.fq.gz ILLUMINACLIP:contams_forward_rev.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36

Please suggest How I can remove barcode and adaptor sequences from raw reads.

rna-seq • 3.1k views
ADD COMMENTlink modified 3.8 years ago by genomax91k • written 3.8 years ago by Bioinfonext250

This question (and some others from past history) make it appear like you do not put time in to understand what you are doing and/or how programs work. While it is tempting to post and get answers here, you are missing out on the larger learning process. Searching the net/looking at past Biostars threads ultimately should get you much more knowledge than posting questions as soon as they come up.

ADD REPLYlink modified 3.8 years ago • written 3.8 years ago by genomax91k

Based on this new question I assume the previous ( How to install Trimmomatic-0.36 ) is resolved. I also linked the manual there, you can find all commands you need, pretty nicely explained too.

ADD REPLYlink written 3.8 years ago by WouterDeCoster44k

Thanks, I able to run Trimmomatic based on your suggestions.

ADD REPLYlink written 3.8 years ago by Bioinfonext250
1

So, therefore, this thread was unnecessary if you would just have spent some effort reading the manual to find the required commands.

ADD REPLYlink written 3.8 years ago by WouterDeCoster44k
2
gravatar for genomax
3.8 years ago by
genomax91k
United States
genomax91k wrote:

Illumina barcodes are never part of the actual read and they are transferred to fastq read header during the demultiplexing. So you never should need to worry about tag/barcodes as long as you are using standard Illumina kits.

You would have to worry about Illumina adapter sequences being present in your reads if your inserts are shorter than the sequencing length (adapter read-through). That is what trimmomatic (and other trimming programs) help you remedy.

ADD COMMENTlink modified 3.8 years ago • written 3.8 years ago by genomax91k

I am using ILLUMINACLIP:contams_forward_rev.fa:2:30:10 as adapter sequence file as you can see in cammand line

There are several adapter sequences, so which sequence should I take in this file.

ADD REPLYlink written 3.8 years ago by Bioinfonext250

If you are actually looking at adapters then trimming programs should be able to scan multiple adapters from that file. I am most familiar with bbduk from BBMap. You only specify one file (adapters.fa included in BBMap distribution) that contains multiple sequences for common commercial kits.

ADD REPLYlink written 3.8 years ago by genomax91k
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