TABLE 2.
Transposon-insertion library preparation in M. abscessus and pooling of ∼200 “rough” colony morphology mutantsa
| Total no. of sequences obtained from Illumina | No. of unique sequences perfectly fitting the predicted pattern of IR-28 bases-IR (sequenced from R1/R2) | No. of unique TA sites mapped to the genome present in both R1 and R2 readings | Total no. of unique mutants identified from the 4 experiments combined |
|---|---|---|---|
| 924,432 | 1,133/1,084 | 73 | 89 |
| 1,142,544 | 2,615/2,377 | 65 | |
| 1,052,922 | 3,569/3,090 | 71 | |
| 1,191,602 | 3,536/2,923 | 77 |
a
The analysis of 200 rough colony morphology clones, isolated from a transposon-mutant library, was repeated four independent times. The procedure used was as follows: (i) gDNA extraction from pooled rough mutants, (ii) MmeI digestion, (iii) blunting, (iv) self-ligation, and (v) PCR and NGS. The analysis resulted in a total of 89 hits (unique TA insertions). The details of the sequencing and analyses and the combined number of identified insertion sites (representing a disrupted gene or affected region between genes) are shown.