Hi colleagues, I need help with MiSeq

[Ivan Castro]

Hi to all, I'm new in this group. I discovered this place thanks to the Nick Loman and Mick Wantson's article http://www.nature.com/nbt/journal/v3.../nbt.2740.html. Friends, I need help with two issues:

- First: I need a software or script for grouping sequences (from a Fasta file) in haplotypes. If anyone know something about this, I will be greatifull with you.

- Second: The next year I will sequence some amplicons with a MiSeq sequencer, but I have never handle a sequence machine. Of course, as biologist I know the Sanger's method, but this machine have advanced tricks for mount the sequence. I need help please, if anyone could explain me how I must mount the sequences I will be greatifull too.
PD: My amplicons have between 400 and 3000 bp.

[gringer]

If you want a strict haplotype signature (no variation permitted), then you can just calculate the MD5 hash (or SHA2, or whatever) of the DNA sequence. With variation it gets a bit trickier, because you need to know which variants are important for haplotyping. A common way is to use variant linkage scores to determine a set of tagging variants that capture the majority of the information, filter on only those variants, then calculate the haplotype hash from that (or more commonly just use the string of variants as your haplotype).

[GenoMax]

Ivan Castro appears to be looking for something simple, along the lines of a multiple sequence alignment to group sequences that are similar. (Creating hashes is an interesting idea (do you have a reference for that?) but the sequences may be of different lengths/not starting at the same position)

For the second question: Are you seeking help with experimental aspects (making the libraries/running the instrument) or are you referring to downstream analysis of amplicons?

@Ivan Castro: It would help if you can describe the aim of your experiment with the amplicons more precisely

[Ivan Castro]

Hi @GenoMax, thanks for reply. The experiment consists on sequencing the 5S rDNA [range length (1,5Kb-3.0Kb)], the 18S rDNA (length 1,8Kb) and the mitochondrial genome (length ~18Kb) which, the last one, will be amplified by the Multiple Displacement Amplification technique. I have read that the Nextera XT kit is the appropriate one for this aim. However, I not understand yet what is the index and the dual indexing and how use this methods for sequencing my amplicons.

About the the haplotypes, I have already wrote a python script for make this task. Obviously, the length of the sequences must be equal.

Thaks for your attention @GenoMax.

[GenoMax]

Here is a tech note from illumina that describes sequencing 16S RNA (which should be similar to your application) on MiSeq: http://res.illumina.com/documents/pr..._miseq_16s.pdf

Nextera XT Kit Sample Prep: http://supportres.illumina.com/docum...15031942_c.pdf

This is a note from Tuft sequencing center that explains how multiplex sequencing works with Illumina: http://tucf-genomics.tufts.edu/docum...q_Adapters.pdf