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Old 06-06-2014, 03:09 AM   #15
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,238
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The question is why short amplicons sequence better than large ones. The evidence is that when a library with broad size distribution is sequenced, after mapping reads, one finds that average size or peak of mapped fragments is smaller than input library indicating preferential sequencing of smaller library fragments. This was not important in earlier days when the libraries were size-selected in a narrow range and multiplexing was not very wide spread. But since introduction of gel free library prep kits (bead based size-selection resulting in libraries with wide distribution of fragment sizes), wide spread use of transposon mediated broad library preps and increased output of platforms it has become more important. When pooling libraries with different insert sizes for sequencing this should be taken into account to obtain desired proportionate number of reads from each library.

My answer as suggested in this thread is “RTA-mediated hypothesis”. Short fragments are more efficient in forming clusters because during bridge amplification it is more likely for polymerase to synthesis a full complementary strand (end to end) for short fragments than large ones due to limited extension time (15 sec in MiSeq). During template generation (early 4-5 cycles) RTA uses signal intensities from images and calls bases from normalised (taking colour cross-talk and phasing correction into account) intensities. Raw data are filtered to remove reads that do not meet signal purity threshold, overlapping and low intensity clusters. At this step in a population of small and large fragment clusters, small ones would have higher intensity (it is proportional to strand number resulting from amplification efficiency) and therefore are preferentially detected and their base composition is called. But large fragments because of less efficient amplification will have less intensity and would not be favoured by RTA. Of course, in a flow cell lane with larger fragments most of the clusters would have less intensity if compared to a lane with predominantly small fragments. But because RTA detection of clusters is relative (normalised intensity not raw), they still are detected and bases are called.

The argument against this is evidence from a large library sequencing (1-5 Kb) in which qPCR predicted cluster density was achieved. I have two arguments against this. Firstly, cluster density and library input are not linear. For example, if 12 pM input gives 800K cluster /mm of a flow cell lane, 8 pM input will not result in 600k cluster. Secondly, quantifying large fragments with KAPA qPCR is not accurate because the standards are 400 bp and their amplification efficiency would be more than large fragments in 1-5 kb range as in this case. In addition, if extension time is not increased significantly, large fragments will drop and only a small portion of library will be amplified and quantified resulting in underestimation of quantity. The attachment in this post is ScreenTape profiles from input library and output from the qPCR reaction showing preferential amplification of smaller fragments during PCR. The qPCR reactions were purified using 1.8x AMPure beads to remove salts, polymerase, SYBR and nucleotides.


Quote:
But SAV doesn't actually depict a cluster any differently that has been recognized by RTA from one that hasn't. Nor does SAV offer any way to verify the hypothesis that short amplicons produce brighter or more robust clusters than long amplicons.
I agree that SAV does not indicate cluster fragment size or which clusters have been selected or passed filter. The pictures I have attached above post is showing that even though image for C channel shows brighter and more clusters than other bases, it has the lowest call for that cycle, swath, tile and surface (image of one spot in one cycle only not average) indicating that RTA has not picked up all possible clusters as RTA-mediated hypothesis predicts.
Attached Files
File Type: pdf Input and qPCR output.pdf (147.9 KB, 15 views)

Last edited by nucacidhunter; 06-06-2014 at 03:15 AM.
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