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Old 11-06-2019, 02:30 PM   #12
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,238
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Quote:
Originally Posted by samd View Post
Hi all,
appreciate the responses

@nucacidhunter
1. Primers are the EMP 515-806 V4 region
2. Cluster density was about 477 if I remember correctly
3. Just 1 library. Usually I was doing 160 samples per run and this time I reduced the run to about 100 samples and still got crummy results
4. Read output was actually 25M and then 23M PF which I guess is great actually but then 40% were undetermined and then when I import the fastq files into QIIME2 I only get about 7 million reads (this is before dada2, so still the entire files).
Run stats seems within specs for the library type although they seem to be more cautious. To increase output safely following can be done:
1- Increasing cluster density to around 800k/mm2
2- Tunning PhiX% to 20 if after mapping undetermind reads majority of them origoinats from PhiX. Undetermind reads also could be reads that their index has not been assigned as a result of miss-matches in custom index primer or PCR primers itself.
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