I'm not able to split libraries from a miseq run where bacteria and archaea libraries were sequenced together. The sequence output is very low. Can anyone suggest if there is a fix for split libraries when amplicon libraries were combined and sequenced. I have used different barcodes
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What kind of libraries are mixed in a pool generally does not matter (except when they are expected to be low nucleotide diversity samples, edit: Which would be the case here since these are amplicons) as long as you have unique barcodes for each sample.
If the sequence output is low (but the samples demultiplexed fine) then that would mean loading more library on a new run may be warranted^^. If you have enough reads but they are not getting demultiplexed (i.e. they end up in "undetermined" reads pool) then that will require a different approach.
Can you clarify which of these two cases we are working with here?
^^ Do you know if a neutral genomic DNA was spiked in (e.g. phiX) on this run? If so at what %? What was the cluster density (K/mm^2) and PF%?
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Input file paths
Most of the sequences were assigned to count of N. Default was set at 0.
Please use
ADD REPLY/ADD COMMENTwhen responding to existing posts to keep threads logically organized.Two relevant lines of interest in your
QIIMElog.There are some reads that have barcodes that are not expected. This does happen and may be acceptable. It is second line that is of more concern.
I am not a
QIIMEuser but I assume that refers to actual reads having N's in them (can you look/post a few reads here). This amounts to almost 70% of your data. That can be due to over-clustering of low-nucleotide diversity sequences, where the basecalling software has difficulty calling bases (where adding neutral genomic DNA becomes essential).I assume you received this data from a sequence provider (can you ask them about genomic DNA %, cluster # and PF%). If there are many N's (software is unable to call a definite base) in the actual reads (or barcodes) this run would have to be repeated. Your sequence provider should not have given you this sequence if there were many N's (unless it was done for diagnostic reasons). Depending on your relationship with the provider it would be best to go back and have a discussion to see what happened and what they may be willing to do. If you had not provided clear information about low nucleotide diversity, then be ready to pay for a re-run.