Nextera and Trimmomatic

Question

[h.l.wong]

Hi all,

I am a newbie here and I have some questions regarding to Nextera sequencing, Trimmomatic and subsequent downstream analysis. I appreciate your help and suggestions. Here is some of the background:

I sent my sequences for Nextera Sequencing and they are paired end sequences. Now I am planning to use Fastqc and Trimmomatic for quality control. Here are the questions:

1. For Trimmomatic, I will use the paired end command so 4 files will be genereated (Forward_Paired, Forward_Unpaired, Reverse_Paired, Reverse_Unpaired). Which files should I choose for downstream analysis (Assembly and binning)?

2. I am planning to assemble the sequences and also go for binning. I know Velvet and Idba are both great programs, which one would you guys suggest? And do both programs take the aforementioned fastq files as input?

Would really appreciate the suggestions and opinions, if my questions are unclear please inform me.

Thanks!

Alan

[Medhat]

what is this the organism? what is the genome size? what is the read/insert length?

for assembly you can use all the data you have

and from velvet manual (you should read it carefully) yes it takes fastq also you need to know

Velvet is a de novo genomic assembler specially designed for short read sequencing technologies, such as Solexa or 454

is this your case?

for Idba

It is a generic purpose assembler and especially good for single-cell and metagenomic sequencing data

is this your case?

[h.l.wong]

Hi Medhat,

It's a mixed community with bacteria and archaea. The seqeuncing platform is NextSeq 500 which the insert length is 200-1000bp.
It's pair ended and the output is 2x150bp fastq files. I have concatenated the files and running Trimmomatic at the moment, which gives me four output (Forward paired, Forward unpaired, Reverse paired and Reverse unpaired).

Should I use the paired files in Velvet?

Thank you for reply

[Medhat]

Yes you should and after that you can evaluate your assembly using tools like Icarus

[finswimmer]

Hello,

For Trimmomatic, I will use the paired end command so 4 files will be genereated (Forward_Paired, Forward_Unpaired, Reverse_Paired, Reverse_Unpaired). Which files should I choose for downstream analysis (Assembly and binning)?

It depends. If you are only interesseted in high quality data you should choose only the the Forward_Paired and Reververse_Paired files. If more coverage is absolutly needed, than the other two files are needed as well, but for this reads you lose the advantages of paired end of course.

fin swimmer