Hi there,
So I submitted 20 RNA samples to a sequencing facility for bulk RNAseq. I got the results back and I actually suspect there might be a mix up of samples. Out of the desperation, I ran a qPCR using a Taqman probe against a well characterised gene and showed that the order of samples are not mixed up (good!) but perhaps the barcodes were wrong. Long story short, how can I confirm if the right barcodes (i5 and i7 combinations from Illumina HT kit) were used for my samples?
To ensure that e.g. RNAseq library #1 were labelled with the right i5/i7 barcode combination, can I do a qPCR on this RNAseq library using the expected i5/i7 combo - if I get a product then its good, if not then that indicates the wrong barcodes were used? Is that even possible?
Thanks heaps! Open to suggestions!
J
Hey there,
may I ask why you think there might be a mix up of samples? So, on what base is your concern?
Eitherway, you might have a hard problem here. I assume you prepared the sample on their own and still have some aliquots left, from before you bulked them? Then you might have the chance to go with your approach, using primers for the barcodes. If you did not prepare the samples by yourself, it might be hard to get the samples, if there are any.
But even if the barcodes got mixed up, you still have unqiue combinations, so your results still should be fine.
Hope this helps a bit. Feel free to ask any further questions.
Regards,
André
Before giving any response, may we know why you initially suspected a mix in the first place? What could have highlighted this? did you have some expectation in from the samples you sent?
Thank you
The easiest is to look into the fastq files. The barcode sequence(s) is a part of the read names in the fastq files. From there you can check the barcode for each sample. If you see the barcodes do not match the samples as you expect you can ask the sequencing facility to give you the configuration file which they used for the sequencing. The config file contains information about the sample number, name and the barcodes. There might just a mixup in the sample names. And then it depends if you prepared the libraries of the facility did it. If you did you have to go back to your notes and make sure the sample names match the barcodes. If the facility made the libraries you have to ask them. If everything fails you can try to do the primer approach on the kept aliquots mentioned above.
If you have any replicates in your samples you can also do a simple expression PCA/MDS plot of the samples and compare the expected clustering (the replicates) with the plots.
Thanks so much for replying Noble Iheukwumere Noble André Marquardt . Here are the details. I compared my qPCR results for a couple of model genes to RNAseq results that were prepared from the same samples. The transcriptional patterns matched but not the sample ID. As I mentioned I got their RNAseq libraries and ran a qPCR to see perhaps maybe the wrong samples were used...but I was able to replicate the transcriptional pattern that I saw using normal qPCR (from samples before RNAseq). Jan Oppelt I did double check the fastq files and all the barcode sequences matched to what was used and there were no unassigned sequences (very little)...suggesting that the right combinations of i5/i7 indices were used but perhaps not in the right samples. I have replicates so I did a PCA/MDS...it was really messy but after re-arranging the sample ID to what I think it might be, I began to see pairs of samples being clustered onto of each other. So this is why I think there might be a mix up of indices being used but off course I was assured that this can't happen by the sequencing facility because they try to automate this process by using multi channel pipettes etc...so my idea to deal with this...is to either do sanger sequencing to identify the barcode in a couple of samples...off course, using RNAseq libraries in sanger sequencing might not work because they are not amplicons...
Anyways, PM me if you want to see data to support this. Thanks heaps!
J
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