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Old 01-21-2014, 03:57 PM   #1
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Location: Australia

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Default MiSeq PE reads R1 failed only R2 worked


I sequenced a pooled library of 22 samples using Nextera library preparation kit provided by Illumina. A 250bp run was performed and a high cluster density was observed during the run, however, after performing quality trimming it was noticed only the singletons generated from R1 was remaining and R2 was exhibiting very low length of only around 30-40 bp. Also the yield for R1 was lower than expected.
Prior to running the library a Bioanalyzer chip was analyzed and a KAPA titration was performed after pooling. The Bioanalyzer chip gave a good curve.
May I please know what could be the reason for this failure? Is it possible that MiSeq is overloaded? Does it something to do with the library prep?

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Old 01-21-2014, 04:17 PM   #2
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What was the cluster density? Were these V3 reagents?

In a case like this it may be best to work with illumina tech support (there is probably local support in your time zone) who will generally do a remote "GoToMyPC" type session to look at your MiSeq, if it is available on the network. Otherwise they will ask you to send some data/log files. Tech support should be able to take a look at the data and help you diagnose the run either way.
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