Optimizing illumina next-generation sequencing library preparation for extremely at-biased genomes
1 Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1SA, UK
2 Oxford Nanopore Technologies, Edmund Cartwright House, 4 Robert Robinson Avenue, Oxford OX4 4GA, UK
BMC Genomics 2012, 13:1 doi:10.1186/1471-2164-13-1Published: 3 January 2012
Additional file 1:
Figure S1. Bioanalyzer quantification and analysis of libraries. Aliquots of the PE-adapter-ligated library (shown) was amplified using enzymes/conditions indicated. After purification with Agencourt Ampure XP beads, library products were analyzed using Bioanlyzer. Bioanalyzer traces show various yields obtained by different conditions and amplification methods used. T7 and PCR-free produced the lowest yield. Phusion and AccuPrime amplifications were undetectable in the presence of TMAC (Tetramethylammonium chloride). Additional file 1, Figure S2. Artemis screen view of coverage. Effect of GC content on coverage for a PCR-free library and four other amplified libraries under test with P. falciparum 3D7 chromosome 1. A) Coverage over the entire chromosome. B) Coverage over high AT-content locus. Kapa HiFi and Platinum pfx libraries shown were amplified in the presence of TMAC. (See Figure 3C for coverage of the GC rich telomere). Additional file 1, Figure S3. Box plots showing coverage analysis of P. falciparum chromosome 11. The effect of TMAC on Kapa HiFi and Kapa2G library amplification. A) Coverage plot for each library on the entire chromosome. Subplots B, C, and D shows coverage of sub-regions of the P. falciparum 3D7 chromosome 11. B) Shows base coverage distribution for each library over gene Pf11_0074 and its neighboring introns. C) Coverage at positions 259985-260864 (extreme AT-region). D) Coverage at positions 29092-30361 (VAR gene and introns). Additional file 1, Table S1. Oligonucleotides used in this study. *Phosphorothioate linkages protect the overhanging thymidine from exonuclease activity. Additional file 1, Table S2. Chimera and duplicate analysis. The average number of chimeric or duplicate reads identified from mapping results is shown for each data set. Percentage of mapped data that were found to be chimeric or duplicated in each data set is shown. Mapped reads were normalized to 21× genome coverage. The two isothermal amplifications generated the highest number of chimeric and duplicate reads. Mapped reads were lower for the duplicate analysis than the chimera analysis because of the different alignment methods used. Additional file 1, Table S3. Ranking analysis. Raw ranking data for P. falciparum 3D7 genome coverage and accuracy scores. Mapped reads were normalized to 21× genome coverage. The number of True positive mismatches, False positive mismatches, Deletion and Insertions identified in each library data sets were used to generate a score before combining all scores to generate an overall mismatch rank. Cov, coverage; MM, mismatch; TP, true positive; FP, false positive; DEL, deletion; IN, insertion. Additional file 1, Table S4. Commercial DNA polymerases used and their catalogue number.
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