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Old 11-08-2017, 07:23 AM   #1
seqtechno1
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Smile strong 140bp peak following RNA library construction

Hello all,
I have created an illumina truseq library on bacteria by first performing ribozero rRNA depletion kit and then performing a truseq stranded mRNA prep while skipping the poly-A selection reaction. my libraries are complete and in cDNA format. i have run a DNA 1000 bioanalyzer assay to visualize the libraries. we see the desired ~260 bp library peak but in every sample there is also a strong ~140bp sharp peak which I think will throw off my molarity calculations (what i'm actually thinking about is proper flow cell cluster density), if it is say, a product of the library prep which cannot form clusters. in certain circumstances it seems to be in much higher concentration than the fragments that i want to sequence. does anyone know what this ~140bp sharp peak is in every sample and i'm also interested to know if there is some way of accounting for this when i do my normalization dilutions pre-pooling. thank you
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Old 11-08-2017, 07:28 AM   #2
HESmith
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The band is adaptor dimers. They form clusters. Use gel or bead purification to remove.
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Old 11-08-2017, 07:36 AM   #3
seqtechno1
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Hi HESmith. thanks for your input. the library construction finishes with two separate ampure xp bead purifications. do you think it is worth performing again?
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Old 11-08-2017, 08:09 AM   #4
microgirl123
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You don't need to perform the bead cleanup/size selection to remove the larger fragments, just the smaller fragments, but you do need to remove them because they will take over a large portion of your sequencing run. You really shouldn't have ended up with so much adapter dimer in the first place - be very careful about adding the correct amount of beads.
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Old 11-09-2017, 02:16 AM   #5
nucacidhunter
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It seems that duel index adapters has been used and clean up has been inefficient in removing excess adapters. The best way will be to tackle it after adapter ligation by reducing clean up bead ratio.

These libraries can be cleaned up one more time. To avoid over clustering due to adapter-dimers you need to use a region covering adapter-dimers as well for average size calculation.
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Old 11-12-2017, 04:55 PM   #6
BioKiwi
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I wonder what brand of beads you have used for clean ups and if they have been used according to TruSeq protocol.
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libary prep, quantitation, ribo-zero, rnaseq, truseq stranded mrna

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