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A paired-end library was constructed, and the average length of insert was about 500 bp.
Moreover, for each fosmid it was consistent across the full length of insert (Table S2B).
The total length of insert sequence generated from the 20 selected fosmids was 708,363 bp, or an average of 35,418 bp per insert.
Uniquely aligned reads were extended 80 bp from the read start site to cover the average length of insert as determined by the Agilent Bioanalyzer.
Additional criteria were also used relative to the predicted length of insert based on human cDNA length, in order to avoid clones of relatively short insert length.
The length of insert cDNA clones ranged from ~600 bp to over 6 kb, which reflected the size distribution of the first-strand cDNA population.
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Interestingly, the insertion mutants display variable length of inserts, ranging from adding 4 to 12 amino acids corresponding to the 8 partially degenerate triples in the designed oligonucleotides.
When we took into account the insertions containing partially assembled sequences, the total length of inserted sequences in Kasalath increased to 7.39 Mb, which, however, was still much less than that of the deleted sequences (13.49 Mb).
To further characterize the mechanisms of donor integration, we sorted the read counts by the length of inserted fragments.
We compared the length of inserted TEs in the genic regions and intergenic regions, and found that the TEs inserted in genic regions are significantly shorter than those inserted in intergenic regions (two-sample Kolmogorov Smirnov (KS) test, P < 0.001) (Supplementary Fig. 11).
For the interior variants, Figure 4 showed the length of inserted peptides for all simulated interior isoforms from rice and Arabidopsis chromosomes.
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