Resequencing

[Julli]

Hey all,
i had a full run in Miseq (250 samples). I work with qiime, after the sequencing i saw i needed to resequence several samples so i entered the run with a colleague who also resequenced some of his samples. Now the problam is that i dont know if the denovo's i get from the second run are the same as the first. so i want to extract only my samples from the second run and then re-do the qiime pipline to make sur each unique read actually gets it own denovo no.
Any suggestions/alternatives?
Thanks

[GenoMax]

Did your samples have unique barcodes? If they did then you should be able to use only your data. If they did not then there is not much you are going to be able to do with this run (if fact your colleague can't use this data either).

[Julli]

GenoMax

Each sample had a unique bacode in our first run. but when we repeated each sample remained with its former barcode (we only have 250 barcodes)

[GenoMax]

Did your samples have unique barcodes (that were independent from your colleague's) when you did the second run? If some of the sample barcodes overlapped then those samples would be non-usable in second run.

[Julli]

Yes. our sample are not overlapping in barcodes

[GenoMax]

Great. If the samples from run 1 and 2 are the same set then you can re-run your Qiime pipeline using the pooled set of sequences leading to a single result.

[Julli]

1. i have the same barcodes from the second run in my first (only 7 samples)
2. Im sorry but i didnt understand what you wrote (im still new in this)

[GenoMax]

Let me see if my understanding is correct.

You have 7 samples that you ran on Run 1. You decided that the number of reads was not enough so you ran the same set of samples a second time. Since the same set of libraries ran a second time (think of it as a technical sequencing replicate) you can merge the sample data sets from the 2 runs into single file for each sample and re-run your Qiime pipeline.

Does this not address your original question?

[Julli]

You undertood correct.
And i understand what you mean, but how can i do that without merging my colleague's samples? (in my first run i have barcodes with the same number?
And, how can i know that for example the qiime give a read the number denovo5 in my 1st run but will give the same sequence denovo70 in the second?

[GenoMax]

Are you working with demultiplexed files or otherwise? If the data is demultiplexed then your samples should have been separated from your colleagues, correct?

[Julli]

How can i know if my data is a demultiplexed files?
I have the Miseq out files (R1,R2,I1) from each sequence run

[GenoMax]

Sounds like the runs are not demultiplexed, if you only have 3 files for each run.

It may be best to demultiplex the two runs so you can separate out your colleague's data from run 2 and then merge the fastq files for each of your samples from the two runs. Demultiplexing can be done using this script: http://qiime.org/scripts/split_libraries_fastq.html

[Julli]

OK. Thank yo very much!