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Old 04-23-2016, 10:06 AM   #1
mlouis
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Default Minimum molecular weight needed for 150 bp PE whole genome resequencing

Hi everyone,

Has anyone any advice about the minimum molecular weight needed for 150 bp PE read Illumina whole genome re-sequencing (coverage of 5x) at BGI?

I am also wondering whether it is better to have high DNA concentration (>200 ng/Ál) and a smearing pattern or lower DNA concentration (20-40 ng/Ál) of mostly high molecular weigth? Samples will be sequenced on an HiSeq X.

Thank you in advance for your help.

Marie
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Old 04-23-2016, 11:36 AM   #2
luc
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What size ranges do your genomic DNA sample fragments have?
Preps for Illumina PE reads are not very demanding with regards to DNA quality - I do not know which protocol the BGI uses; better ask them.

BTW, does the BGI really run a HiSeq X sequencer?
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Old 04-27-2016, 08:15 AM   #3
mlouis
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Thanks for your answer. The range is 3 to 10kb for most of them but for some it is 250 bp - 10 kb.

Yes they run a HiSeq X.

Best,

Marie
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Old 04-27-2016, 10:19 AM   #4
cliffbeall
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Illumina sequencing doesn't work well on fragments over ~1000 bp because the cluster amplification is less efficient for longer pieces. You can use a Covaris, BioRupter or something similar to reduce the size if your DNA is longer. You wouldn't want very short fragments, say under 200 bp because the sequence reads will go into the other adapter too often. Either concentration range you mention should be fine.
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Old 04-27-2016, 04:15 PM   #5
luc
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Library prep and sequencing is possible with this type of somewhat degraded sample. However you might introduce some more biases than usual and your library might show a double-hump or another unusual size distribution in the bioanalyzer. Thus. a good library size selection might be necessary.
You can sonicate the samples according to standard protocols.
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Old 04-30-2016, 05:31 PM   #6
LOH
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You can shear your DNA samples into 300-500 bp size range according to standard protocols. Over 500 bp size is not good for sequencing on illumina platform. http://support.illumina.com/content/...15026486_c.pdf
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Old 05-15-2016, 03:25 AM   #7
mlouis
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Thanks for your answers.
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