Hi SEQ-users,
We are currently following the Illumina Demonstrated Protocol for 16S sequencing on the MiSeq (24-96 samples) for stool & saliva samples. We are experiencing some inconsistencies with the results of the run metrics (e.g. %Q30 ranges between 64 - 85%, cluster density between 421 to 1328 K/mm2 etc) between each run.
I was just wondering for those who use the same protocol,
1. What does your 16S MiSeq sequencing run look like? (in terms of %Q30, cluster density, % aligned etc).
Have you set specific run metrics for run acceptance?
(I have attached the run summary of our recent run, let me know your thoughts.)
2. What is the minimum number of sequences you process per sample?
Apart from the Illumina document, do you know any publication that recommends a certain number of reads per sample?
3. We are using USEARCH for quality filtering before assembly. However we get very low R2 reads. Would you recommend other quality filtering tools?
Any answers or suggestions would be greatly appreciated.
Thank you in advance.
We are currently following the Illumina Demonstrated Protocol for 16S sequencing on the MiSeq (24-96 samples) for stool & saliva samples. We are experiencing some inconsistencies with the results of the run metrics (e.g. %Q30 ranges between 64 - 85%, cluster density between 421 to 1328 K/mm2 etc) between each run.
I was just wondering for those who use the same protocol,
1. What does your 16S MiSeq sequencing run look like? (in terms of %Q30, cluster density, % aligned etc).
Have you set specific run metrics for run acceptance?
(I have attached the run summary of our recent run, let me know your thoughts.)
2. What is the minimum number of sequences you process per sample?
Apart from the Illumina document, do you know any publication that recommends a certain number of reads per sample?
3. We are using USEARCH for quality filtering before assembly. However we get very low R2 reads. Would you recommend other quality filtering tools?
Any answers or suggestions would be greatly appreciated.
Thank you in advance.
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