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Global fitness profiling of fission yeast deletion strains by barcode sequencing

Tian Xu Han, Xing-Ya Xu, Mei-Jun Zhang, Xu Peng and Li-Lin Du*

Author Affiliations

National Institute of Biological Sciences, 7 Science Park Road, Zhongguancun Life Science Park, Beijing, 102206, PR China

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Genome Biology 2010, 11:R60  doi:10.1186/gb-2010-11-6-r60

Published: 10 June 2010

Additional files

Additional file 1:

Information on barcode decoding by deep sequencing.

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Additional file 2:

Diagrams of the two methods used to decode barcodes. (a) Paired-end deep sequencing. (b) Smart pooling and multiplexed deep sequencing.

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Additional file 3:

The barcode sequences uniquely associated with the mutants in Bioneer version 1.0 haploid deletion library. Column A, gene name. Column B, well position according to information provided by Bioneer. Column C, well position annotation: W denotes wrongly placed strains that have been located to a different well by the smart pooling data and PCR analysis (also separately listed in Additional file 5); M denotes the strains that are indicated by deep sequencing to be present in more than one well (also separately listed in Additional file 6); C denotes the wells that are indicated by deep sequencing to be contaminated by a different strain. Column D, uptag sequences. Column E, dntag sequences.

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Additional file 4:

Barcodes used by more than one deletion strain. These barcodes cannot be assigned to unique strains and are not included in Additional file 3. Some of the barcodes listed here have been verified by Sanger sequencing (two examples are shown in Additional file 7).

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Additional file 5:

Strains whose well positions differ from information provided by Bioneer (annotated with the letter 'W' in Additional file 3). These strains have all been individually verified by PCR analysis (examples shown in Additional file 7).

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Additional file 6:

Strains present in more than one well (annotated with the letter 'M' in Additional file 3). The well positions are predicted by the smart pooling data. The two wells harboring the same strains are often not immediately adjacent wells, and many of them are not even in the same 96-well plates, suggesting that most of the cross-contaminations probably had happened before we received the library from the supplier. Some of the contaminated wells have been verified by PCR analysis (examples shown in Additional file 7).

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Additional file 7:

Experimental verification of barcode sequences and strain locations revealed by deep sequencing. (a) Sanger sequencing of deletion cassettes sharing the same barcodes. (b) PCR analysis of misplaced strains and those present in more than one well.

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Additional file 8:

The linearity and dynamic range of barcode sequencing assessed using spike-in controls. A rad32 deletion strain and a rad26 deletion strain from the Bioneer version 1.0 upgrade package (M-1030H-U) were spiked into 24 version 1.0 pooled samples that had been grown in minimal or rich medium for different generations. The ratios between the cell number of each spike-in strain and the total cell number of the version 1.0 pooled strains were 1/200, 1/1,000, 1/2,500, 1/5,000, 1/10,000, and 1/20,000. The read numbers were normalized by total matched reads of the version 1.0 strains. (a) The normalized read numbers were plotted against the spike-in ratios. (b) The observed log fold changes between different spike-in samples were plotted against expected log fold changes.

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Additional file 9:

The GI values of mutants grown in rich versus minimum medium (YES versus EMM).

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Additional file 10:

The GI values of mutants grown in lysine supplemented minimal medium versus minimum medium (EMM+K versus EMM).

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Additional file 11:

The GI values of mutants treated with TBZ, CPT, HU, and UV.

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Additional file 12:

A list of 68 TBZ-sensitive mutants and their GI values.

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Additional file 13:

A list of 113 CPT-sensitive mutants and their GI values.

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Additional file 14:

A list of 23 HU-sensitive mutants and their GI values.

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Additional file 15:

A list of 38 UV-sensitive mutants and their GI values.

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Additional file 16:

Comparison of the Deshpande et al. CPT screen hits with our profiling results.

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Additional file 17:

Comparison of the Deshpande et al. HU screen hits with our profiling results.

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Additional file 18:

The full heat map of the hierarchical clustering analysis shown in Figure 4e.

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