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Old 01-29-2012, 04:00 PM   #1
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Location: Sydney, Australia

Join Date: Jun 2011
Posts: 163
Default Typical Quantity of Primer Sequences In Final Library


I'm wondering what amount of contamination is typical of a multiplexed library ? We've just done our first ever multiplexing run through a service provider on the HiSeq for a number of ChIP-seq experiments against histone mark H2A.Z. Here is FASTQC output for one sample :

Basic Statistics
Total Sequences 35111957
Sequence length 51

Overrepresented sequences
Sequence Count Percentage Possible Source
ATCGGAAGAGCACACGTCTGAACTCCAGTCACCAGATCATCTCGTATGCCG 2115847 6.026001341936025 Multiplexing_PCR_Primer_2.01 (100% over 32bp)
GAGCACACGTCTGAACTCCAGTCACCAGATCATCTCGTATGCCGTCTTCTG 1385332 3.945470769402002 PCR_Primer_Index_7 (100% over 39bp)
AGAGCACACGTCTGAACTCCAGTCACCAGATCATCTCGTATGCCGTCTTCT 910631 2.5935068216220474 PCR_Primer_Index_7 (100% over 38bp)
GGAAGAGCACACGTCTGAACTCCAGTCACCAGATCATCTCGTATGCCGTCT 413853 1.1786668569911953 PCR_Primer_Index_7 (100% over 35bp)
GAAGAGCACACGTCTGAACTCCAGTCACCAGATCATCTCGTATGCCGTCTT 177781 0.5063260928463771 PCR_Primer_Index_7 (100% over 36bp)
AGCACACGTCTGAACTCCAGTCACCAGATCATCTCGTATGCCGTCTTCTGC 169413 0.48249375561721036 PCR_Primer_Index_7 (100% over 40bp)
AAGAGCACACGTCTGAACTCCAGTCACCAGATCATCTCGTATGCCGTCTTC 154312 0.4394856145443559 PCR_Primer_Index_7 (100% over 37bp)
CGGAAGAGCACACGTCTGAACTCCAGTCACCAGATCATCTCGTATGCCGTC 128319 0.36545670182952206 PCR_Primer_Index_7 (100% over 34bp)
GATCGGAAGAGCACACGTCTGAACTCCAGTCACCAGATCATCTCGTATGCC 117745 0.33534160457077344 Multiplexing_PCR_Primer_2.01 (100% over 33bp)
GCACACGTCTGAACTCCAGTCACCAGATCATCTCGTATGCCGTCTTCTGCT 94592 0.2694011045866797 PCR_Primer_Index_7 (100% over 41bp)
ACACGTCTGAACTCCAGTCACCAGATCATCTCGTATGCCGTCTTCTGCTTG 47410 0.1350252280156301 PCR_Primer_Index_7 (100% over 43bp)

Also, are there any protocol tricks so that tags aren't being wasted just sequencing primers ?

I realise there's also the problem of overamplification here (3.5 million unique mapping reads with Bowtie). I hear that happens often when indexes are used ?
Dario1984 is offline   Reply With Quote
Old 03-05-2012, 07:44 AM   #2
Location: Boston, MA

Join Date: Apr 2011
Posts: 49

It sounds like you're doing ChIP Seq... are you using the TruSeq kit? If so, I can tell you what we do. I use 10 to no more than 50 ng of input as measured by pico green, and a 1:50 dilution of the adapters. Usually 15-18 cycles of PCR are required. I also do the size selection AFTER the PCR, which removes any dimer.
jlove is offline   Reply With Quote

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