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Old 02-24-2019, 10:15 PM   #1
BAGeno
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Unhappy Illumina NextSeq shorter fragment size than expected

Hi,

We were preparing library for NextSeq whole exome sequencing. Our starting concentration before library preparation for three samples were 27ng/ÁL, 37ng/ÁL and 42ng/ÁL. We pooled library and when we checked our fragment size before running on sequencer by Bioanalyzer, we saw fragment size of 239 base pair which is smaller than expected. I want to know on which step we are making mistake which resulted in smaller insert size than expected?
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Old 02-24-2019, 10:37 PM   #2
nucacidhunter
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If you can post Bioanalyser result then it will be easier to diagnose the issue.
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Old 03-06-2019, 09:45 PM   #3
BAGeno
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Default Bioanalyzer image

https://drive.google.com/file/d/0ByA...ew?usp=sharingWe have repeated procedure of tagmentation and we have got same results of shorter fragment length
This is the image of bioanalyzer peak.
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Old 03-07-2019, 12:31 AM   #4
nucacidhunter
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The fragment size distribution is within optimal for sequence capture if that is what you are doing. To obtain larger fragments (if you are using Nextera not Nextera flex) input DNA need to be increased. If you are comparing the library size with Illumina user guide the difference could be also due to Bioanlyser assay (note Illumina have used HS DNA Chip).
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Old 03-07-2019, 01:29 AM   #5
BAGeno
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This is size of library after adding adapters. Is 200-400bp normal? This will affect concentration or not?
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Old 03-07-2019, 09:42 AM   #6
itstrieu
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Which library prep and capture kit are you using?
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illumina 1.8, nextseq 500, read length

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