'Hallo' from Germany!

[Paralia]

Hi all!

I'm a PhD-student in Germany and work with small eukaryotes that thrive within ocean currents (Plankton). In one of my projects we sequence (Illumina Miseq) amplicons from more than 50 samples to gain a picture about the diversity of protists in the water column.

Since I haven't work with Illumina before, I have a plenty of questions for which I hope to find answers in this forum.

One urgent question (and stupid I guess.. as it is always :
Can you work with paired-end reads when only one Primer (Reverse) was labeled? Is a correct assembling of the reads and their assignment to the different samples possible? Even if only read1 can be related to one sample, it doesn't matter were read2 originates, or? .. The main thing is, that there is enough overlapping (e.g. 90 bp) and no reads are left.. or am I totally wrong?

I am confused... hope you can help!

Paralia

[AStretton]

What do you mean only one primer was labeled? Do you mean only the reverse primer contained the index?

[Brian Bushnell]

Read 2 always originates from the same molecule as read 1.

[Paralia]

Thanks for your answers!

We have reverse primers which are labeled with 1-4 N's and 20 different Hexamers to discriminate between the samples and sended them to a sequencing company which then did the adapter ligation, indexing ect.. We achieved 2 x 250 bp. However, we ordered single end and got paired end and now I am wondering if I can work with the paired end, even if only one read (with the labeled rev. primer) can be attributed to a single sample.

I hope you understand my problem...

[AStretton]

Ok if the company did separate indexing to your labelled primers then you can use both read 1 and read 2, when it is sequenced the index essentially labels the cluster from which both read 1 and read 2 come from so they are always linked. (I am assuming the sequencing was done on Illumina sequencers here)

Bottom line the answers is yes you can use both read 1 and 2.

[Paralia]

Ah.. okay! Then I guess we have saved a lot of money because we got more than we ordered.

Thanks again!