Figure 1.
Overview of the two sequencing methods used in this study for sequencing of large-insert cosmid clones.
Traditional barcoded sequencing (left) uses DNA barcodes to keep clones as separate samples throughout the sequencing and assembly process. Pooled sequencing (right) involves combining clones into one sample for sequencing and assembly, and subsequently using previously obtained Sanger “end-tags” to retrieve specific clone sequences.
Table 1.
Metagenomic and genomic libraries used in this study.
Figure 2.
Alignment identity between pooled sequencing result and barcoded sequencing result.
For all 73 clones, end-tags were used to retrieve contigs from pooled sequencing results; retrieved contigs were aligned to the reference barcoded sequencing result, and clones were binned by percent identity.
Figure 3.
Percent coverage of pooled sequencing result relative to barcoded sequencing result.
Each of the 73 clones was categorized into Clone Types A, B, C, or D by the number of end-tags obtained (one or two), whether the end-tag retrieved a contig from the pool, and the completeness of the retrieved pooled sequencing result relative to the reference barcoded sequencing result (full or partial coverage). Clone Type descriptions are given above.
Figure 4.
Retrieved coverage and estimated actual coverage of pooled sequencing relative to barcoded sequencing.
(A) An example clone, Lactose clone 20, shows retrieved coverage at 48% (using end-tags as queries), but an actual coverage of 98% (using barcoded result as query). (B and C) Percent coverage for each of the 73 clones, binned in ten-percent increments. Retrieved coverage (B) is compared to estimated actual coverage (C).
Figure 5.
Heat map of clone sequence similarity and corresponding bar plots of clone coverage.
Pair-wise sequence similarity is shown for all 73 clones (A), juxtaposed to their pooled sequencing coverage, showing both retrieved coverage (B) and actual coverage (C).
Figure 6.
Overlapping clones assemble into one contig.
Three overlapping clones as revealed by barcoded sequencing (above) and pooled sequencing (below). Locations of end-tags are indicated by vertical dashed lines. White dashed boxes indicate gaps in the pooled sequencing data; black boxes indicate a contig. Lengths of all contigs are given.