[Sarutis]

Thanks, relipmoc
Since you were so kind to answer quickly, I'll ask few more things:
what do you use to connect overlapping paired sequences? I found PEAR and stitch as avaliable softwares, but I'd like an expert opinion

[relipmoc]

Skewer supports demultiplexing amplicon pairs

I'm new to MiSeq sequencing and to this blog and I'm having difficulties:

I'm sure these info are somewhere in the blog, but would someone be so kind and tell me where to find the primer design info for barcoded amplicon library for a 2x300 MiSeq run?
Are there any other info needed
Thanks in advance

Recently we indexed 6 amplicon libraries and sequenced them in a 2 x 300bp MiSeq run. Each of these 6 amplicon libraries was also a mixed library which contains about 60 samples barcoded by different combination of forward-primer sequence and reverse-primer sequence (In fact, fragments from these samples were also ligated with 6 nucleotides index sequences). Hence we may demultiplex about 360 samples using this protocol.

In my understanding, the first level barcoding is easy to handle, you should note down the 6 bp index sequences when construct the libraries, and later you can specify the 6 bp sequences in the Sample Sheet when preparing a MiSeq run. For the second level barcoding, you need to know what gene region is amplified with what primers (forward and reverse).

For demultiplexing, I suggest you use skewer (http://sourceforge.net/projects/skewer/) which supports demultiplexing amplicon pairs.

[Sarutis]

2x300 MiSeq amplicon libraries barcoded primers

I'm new to MiSeq sequencing and to this blog and I'm having difficulties:

I'm sure these info are somewhere in the blog, but would someone be so kind and tell me where to find the primer design info for barcoded amplicon library for a 2x300 MiSeq run?
Are there any other info needed
Thanks in advance

[nucacidhunter]

Yes. They are perfect match if prepared by standard method. Both would have relatively similar peak size, so preferential clustering of smaller fragments will not be an issue and you can expect to get reads from both in proportion to input library.

[Katerina Cvikova]

Hi everybody,
do you know if it is possible to sequence in one run on MiSeq libraries from Nextera DNA and TruSeq PCR-Free together?

[ramkrissh]

Can any body has details of illumina miseq sequencing for 16S amplicon library construction specific to V3 region only . I need primer , adapter and barcode sequences used for V3 region 16S sequencing ..... Please help

[shirley971002]

PCR primers sequences?

The original adapters (what I called legacy) released by Illumina are not exactly the same sequence as the currently in use TruSeq adapters - e.g. old adapters did not have indexes TruSeq ones do. The sequences for the individual TruSeq adapters can be found in the Illumina customer letter (see attached) which can also be downloaded from the Illumina website. I have also attached an alignment I had illustrating the differences between the original adapters and the currently in use TruSeq adapters.

sorry,I am new.I want to know, in Truseq vs old adapters.pdf, which are PCR primers? Can you give the specific sequences? Thank you !

[jngao1204]

Thanks for sharing!

[pmiguel]

According to my calculation there will not be any overlapping. Plz correct me if i am wrong.
Thanks!

That would depend on the length of the reads.

--
Phillip

[bssharma]

I am sure you can do the calculation yourself.

--
Phillip

According to my calculation there will not be any overlapping. Plz correct me if i am wrong.
Thanks!

[pmiguel]

Hi, I think i got my answer, thanks. If you can also tell me in Illumina sequencing which sequencing read length format generates overlapping end? and how long is the overlapping region for 230bp insert?
Thank you so much

I am sure you can do the calculation yourself.

--
Phillip

[bssharma]

230 bp? (350 - (2 x 60))
Not entirely clear what you are asking, though. I am presuming by "350 bp DNA fragment" you mean, 350 bp amplicon.

Hi, I think i got my answer, thanks. If you can also tell me in Illumina sequencing which sequencing read length format generates overlapping end? and how long is the overlapping region for 230bp insert?
Thank you so much

[bssharma]

Hi, I think i got my answer, thanks. If you can also tell me in Illumina sequencing which sequencing read length format generates overlapping end? and how long is the overlapping region for 230bp insert?
Thank you so much

[pmiguel]

Can anyone plz tell mE what is the insert length (bp) (assuming adaptor length is 60bp on each end) for 350bp DNA fragment subjected to paired end sequencing.
THANKS!

230 bp? (350 - (2 x 60))
Not entirely clear what you are asking, though. I am presuming by "350 bp DNA fragment" you mean, 350 bp amplicon.

--
Phillip

[bssharma]

Insert length?

Can anyone plz tell mE what is the insert length (bp) (assuming adaptor length is 60bp on each end) for 350bp DNA fragment subjected to paired end sequencing.
THANKS!