Variable Copy Number Tandem Repeats Have Been Observed in a Number of Prokaryotic and Eukaryotic

Supplementary material:

Supplementary Fig. 1: Lack of correlation between ETANDEM score and size variability in ORFs containing a leader sequence but lacking a GPI anchor. The TR region of the 10 top-scoring ORFs containing (A) a leader sequence but lacking a GPI anchor or (B) lacking both a leader sequence and GPI anchor was amplified by PCR in 11 clinical A.fumigatus isolates. (A) Afu6g06670, Afu2g03540, Afu5g06880 (ETANDEM scores 113, 88 and 75, respectively) are size variable. For unknown reasons, the TR region of Afu7g08500 (ETANDEM score 250) was amplified in only two isolates (isolate 8 and Af293). (B) Afu7g08290, Afu7g08310, Afu6g09340, Afu8g06150, Afu6g09360, Afu5g15090, Afu7g07060 (ETANDEM scores 670, 400, 388, 232, 226, 218 and 190 respectively) are size variable. For unknown reasons Afu7g07100 (ETANDEM score 1032) was amplified only in Af293.

Supplementary Fig. 2: Tandem repeat regions in GPI anchor and leader-sequence-containing size-variable genes encode repetitive amino-acid blocks highly enriched with serine, threonine and proline amino-acid residues. The cDNA sequences of the size-variable TR regions in Afu3g08990, Afu2g05150, Afu4g09600 and Afu6g14090 (A-D, respectively) contain different repeat numbers among A.fumigatus isolates. TR regions in these genes are consistent with the ORF encoding conserved amino-acid blocks (Afu3g08990: P-G-Q-P-S-A/V; Afu2g05150: E-T-S-T-P-C-E-T-T-T-T-P/T-T; Afu4g09600: G-F-H/R-K-R-G-G-G-D-T-T-V-I-G-G-P-S-G-D-D-G-G-N-S-A-x(0,2)-E-x(0,2)-F-D/E-S-S/T-Y-A/E-S-S-V-K-D-W/Y-Y-K-D-D-H-S-V-D; Afu6g14090 : G-S).

Supplementary Fig. 3: The Afu3g08990-deleted strain shows wild-type (Af293) levels of virulence in a disseminated model of invasive aspergillosis. Survival curves of mice intravenously injected with an inoculum of 2.5 x 105 conidia/mouse of the wild-type Af293 strain (n=12 mice), and Afu3g08990-deleted D1 strain (n=12 mice). Percent survival was monitored daily over the 21-day study period.

Supplementary material

Supplementary Table 1: Primers used in this study

Gene name / 5’ primer sequence / 3’ primer sequence
Non-leader
Afu7g07100 / AACTTGCAATACAGTTGCATATATTGGTTGGACCAT / CACATTATTGCCACTGGTAACCGTACATGT
Afu7g08290 / TGGAGTCCAGAATTGCAAACCTTG / ATAATATCCCAGGTAGGTAATCTGCTG
Afu7g08310 / AAGCTGGACCGTCCACACCTCTTCATGGAC / CGAACCAAAAGCCCCTGCTCAGTTTCCAC
Afu6g09340 / ATGCGGACTCCAGATATCAGAAATGATACTATT / CAAGAAGTGCGACCACACCAAGAAGTGTGGACAA
Afu4g10350 / GCCGCACTCTTTCCGATTATAACATCCAGAAG / TAGCATCCACCACGCAGACGCAGCACCAGGT
Afu7g08240 / CTGGTGACTTCAACCTGCAACC / CTAACTGCCGATGAAGTACCATG
Afu8g06150 / GCTGCTTCTAGTTCTCTCAATCC / CTCACTGGACTGACAATTAGAGG
Afu6g09360 / TTGTCTGGAAGGTGAGAGAGACT / AATGGCGTGGCACCCATATCAAA
Afu5g15090 / GGTATGGTATGTCTGGCTGGTGA / CGGCTGATGCTGATGGCGATG
Afu7g07060 / AGACGACACCGACAGCATGGAT / CAGCTCGTCTTCATCTGACTCTG
Leader
Afu1g01020 / AGGGAGCAGATGTCAATGTCTGTG / CATTCAGCTGTTGAGGAAAGCATCC
Afu2g18090 / ATGGATGCTGGCTGCAAAATGAA / TAGGAGGGGCAGGAGGTGGAT
Afu7g08500 / CCTCCATGTGAATGAGAGACTGAC / CTCTTTCACCTCGGAAGCATACCCAAGAACTCTCAAC
Afu3g07400 / GCCATTGGGGCATTGGAGAGAGTTCCGCCTCA / TGGACCGCAATCGGAGTCCTTGTGGCACAC
Afu6g06670 / GATTCAAAAGCGGCAGATGAAGGAGAAG / CTTTGAACCAACTCTTGAGTCTA
Afu2g03540 / TGCCACTCGCAGCAGCAGCATGGATGCTACTC / GTTGGTGCAGCAGGAGTGGGACATGGTGGA
Afu8g00630 / AATACATCGTCAGCGAAATAGACC / GCTTGCAGTGGCCTTGAAATGAC
Afu5g06680 / GGTTGTCAACATCTACTCTGAGG / CGTTATGCGTGAAGGCTCTGCT
Afu8g07170 / TTGCCATTCTCTCGGCTGTGTG / CATCCTTGACATATTTGTCAAAGC
Afu5g11300 / ACCTTCTCGCTGTGCAGGACTT / GTGCTAATGCGATTGCGAAGTCT
GPI +Leader
Afu3g08890 / TAGTACAATCACTGTGACTACCAC / ATGACAACCCAGATACCAGCCAG
Afu2g05150 / TCGAACTTGGCTACGACCCTGTCTGGCAAG / GCAGGAGGTGTAGTGGGTGTGGTGGTGGTT
Afu4g09600 / AAGGACGCGTACACGGACGAC / CTGCTTATGGGAGTTTGGAGTGC
Afu6g14090 / GATAAATGTCTCTGCTCCCAAGCC / CTAAAGGATAATCAAGGCAGCGAG
Afu4g03240 / TCTCCAAGAACGACAAGTTCGTCG / TTAGAGAGCGACGGCGATGGC
Afu7g00970 / CATCGGGGAGATCTGTACTGGAC / CAGCACCCACGCCGGCGTTC
Afu3g00270 / CCCAGTACGGAGATGATTTGGCTAAGC / CTCAGGATGTTGGAAGTTGAAGGAGCA
Afu1g16190 / CAAGGATGCCGTTACCTGGTCTATT / TTAGAATGCCAACACGGCAGCGACGG
Afu3g00880 / ATCCCTCCTCCTTCGACATCTACCTCGTCAAGA / TTACAGAGCCAGGGCGACCAGACCAA
Afu2g07800 / GTGCAACAAGTGGTATGTCTTGTC / CTACAGCAGGTAAGCAGCCAGG
General
gpdA / TCTCCAACGTTCTTGCACC / CCACTCGTTGTCGTACCAGG
MDR1 / TCAAGGCTCGTGACGACGTTACT / CTCAGTCATGTACTTCTTGGCGG
AscI-08990
Hyg
08990-S
08990 prom
08990 myc / ATGGCGCGCCAGGTAGCGGAATGGAAATAGACG
ATGGTACCGTCGACAGAAGATGATATTGAAGG
GCATCGCACACCCATGCGTGA
GGCCGGCCGTTGACGATGGGCA
GTGGGATAA
GCGGCCGCGGCGGCCAAAAGCTCATTTCTGAAGAGGACTTGGGCGGCCAAAAGCTCATTTCTGAAGAGGACTTGAACAAGGGCAATGACGAGGTAT / ATGGCGCGCCGTAAGGACAGACACCACCTGAC
ATCACGTCGCTCTCCCTTATGCGACTCCTGCA
CTGATACAAGTCACTCTCGTTCC
GCGGCCGCTGGCACAATGCCACCCAAGGCT
GGCGCGCCGAACTATACCGTCAAAACCTTTTC

Supplementary Fig. 2.

A) Afu3g08990

i. Af293. 32 repeats

GTTGCCCCTGGACAGCCTACAGCTCCTAGCCAGCCTTCTGTCCCTGGTCAGCCCTCTGTTCCTGGCCAGCCTTCTGTCCCTGGTCAGCCCTCTGTTCCAGGCCAGCCTTCTGTTCCTGGCCAGCCCTCTGTTCCCGGCCAGCCTTCTGTTCCCGGCCAGCCCTCCGTTCCCGGCCAGCCTTCTGTTCCCGGCCAGCCCTCTGTTCCCGGCCAGCCCTCTGTTCCCGGCCAGCCTTCTGCTCCCGGCCAGCCTTCTGCTCCCGGCCAGCCTTCTGTTCCTGGCCAGCCTTCTGTTCCCGGCCAGCCCTCTGTTCCCGGCCAGCCTTCTGTTCCTGGCCAGCCCTCCGTTCCCGGCCAGCCTTCTGCTCCCGGCCAGCCTTCTGTTCCTGGCCAGCCTTCTGTTCCCGGCCAGCCCTCTGTTCCCGGCCAGCCTTCTGTTCCTGGCCAGCCCTCCGTTCCCGGCCAGCCTTCTGTCCCTGGTCAGCCATCTGCGCATGAGCAGCCTTCTGTTCCTGGCCAGCCCTCTGTGCATGAGCAGCCATCTGATCCTGAGCAGCCATCTGTTCATGGTCAACCCACTGCCGTATCG

VAPGQPTAPSQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSAPGQPSAPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSAPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSAHEQPSVPGQPSVHEQPSDPEQPSVHGQPTAVS

ii. AF10. 25 repeats

GTTGCCCCTGGACAGCCTACAGCTCCTAGCCAGCCTTCTGTCCCTGGTCAGCCCTCTGTTCCTGGCCAGCCTTCTGTCCCTGGTCAGCCCTCTGTTCCAGGCCAGCCTTCTGTTCCTGGCCAGCCCTCTGTTCCCGGCCAGCCTTCTGTTCCCAGCCAGCCTTCTGTTCCCGGCCAGCCTTCTGTTCCCGGCCAGCCTTCTGTTCCCGGCCAGCCCTCTGTTCCCGGCCAGCCTTCTGTTCCTGGCCAGCCCTCCGTTCCCGGCCAGCCTTCTGTTCCCGGCCAGCCCTCTGTTCCCGGCCAGCCTTCTGCTCCCGGCCAGCCTTCTGCTCCCGGCCAGCCTTCTGTTCCTGGCCAGCCTTCTGTTCCCGGCCAGCCCTCTGTTCCCGGCCAGCCTTCTGTTCCTGGCCAGCCCTCCGTTCCCGGCCAGCCTTCTGTCCCTGGTCAGCCCTCTGCGCCTGAG

VAPGQPTAPSQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPSQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSAPGQPSAPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSAPE

iii. AF9. 18 repeats

GTTGCCCCTGGACAGCCTACAGCTCCTAGCCAGCCTTCTGTCCCTGGTCAGCCCTCTGTTCCTGGCCAGCCTTCTGTCCCTGGTCAGCCCTCTGTTCCAGGCCAGCCTTCTGTTCCTGGCCAGCCTTCTGTTCCCGGCCAGCCTTCTGTTCCCGGCCAGCCTTCTGTTCCTGGCCAGCCCTCCGTTCCCGGCCAGCCTTCTGCTCCCGGCCAGCCTTCTGCTCCTGGCCAGCCTTCTGTTCCCGGCCAGCCCTCTGTTCCCGGCCAGCCTTCTGTTCCTGGCCAGCCCTCCGTTCCCGGCCAGCCTTCTGTCCCTGGTCAGCCCTCTGCGCCTGAG

VAPGQPTAPSQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSAPGQPSAPGQPSVPGQPSVPGQPSVPGQPSVPGQPSVPGQPSAPE

B) Afu2g05150 MP-2

i. Af293 18 repeats

CCTACCGAGACCTCCACTCCGTGCGAGACTACTACCACTACTACCGAGACCTCTACCCCCTGTGAGACTACTACTACGACTACTCCTCCTCCTGAGACCTCCACTCCGTGCGAGACTACTACCACTACTACCGAGACCTCCACTCCATGTGAGACCACTACCACTCCTACCGAGACCTCTACCCCCTGTGAGACTACTACTACCCCTACCGAGACCTCTACTCCGTGTGAGACCACTACCACTCCTACCGAGACCTCTACCCCCTGTGAGACTACTACCACTACTACCGAGACCTCTACCCCCTGTGAGACTACTACCACTCCTACCGAGACCTCTACCCCCTGTGAGACTACTACTACCCCTACCGAGACCTCTACTCCGTGTGAGACCACTACCACTCCTACCGAGACCTCTACCCCCTGTGAGACTACTACCACTACTACCGAGACCTCTACCCCCTGTGAGACTACTACTACGACTACTCCTCCTCCTGAGACCTCCACTCCGTGTGAGACCACTACCACTCCTACCGAGACCTCTACTCCATGCGAGACTACCACCACTCCTACCGAAACCTCTACCCCCTGTGAGACTACTACTACGACTACTCCTCCTCCTGAGACCTCCACTCCGTGCGAGACCACTACCACTCCTACCGAGACCTCTACCCCCTGTGAGACTACTACTACCCCTACCGAGACCTCTACTCCGTGTGAGACTACTACTACTCCTCCTGAAACC

PTETSTPCETTTTTTETSTPCETTTTTTPPPETSTPCETTTTTTETSTPCETTTTPTETSTPCETTTTPTETSTPCETTTTPTETSTPCETTTTTTETSTPCETTTTPTETSTPCETTTTPTETSTPCETTTTPTETSTPCETTTTTTETSTPCETTTTTTPPPETSTPCETTTTPTETSTPCETTTTPTETSTPCETTTTTTPPPETSTPCETTTTPTETSTPCETTTTPTETSTPCETTTTPPET

ii. AF31. 9 repeats

PTETSTPCETTTTTTETSTPCETTTTTTPPPETSTPCETTTTTTETSTPCETTTTPTETSTPCETTTTPTETSTPCETTTTPTETSTPCETTTTPTETSTPCETTTTPTETSTPCETTTTTTPP

iii. AF36. 11 repeats

CTACGACTACTCCTCCTCCTGAGACCTCCACTCCGTGCGAGACCACTACCACTCCTACCGAGACCTCTACCCCCTGTGAGACTACTACTAGCCCCTACCGAGAG

PTETSTPCETTTTTTETSTPCETTTTTTPPPETSTPCETTTTTTETSTPCETTTTPTETSTPCETTTTPTETSTPCETTTTPTETSTPCETTTTPTETSTPCETTTTPTETSTPCETTTTTTPPPETSTPCETTTTPTETSTPCETTTSPYRE

C) Afu4g09600

i. Af293. 3 repeats

CATCTCGCGGTTTCCATAAGAGAGGTGGGGGTGACACCACAGTCATTGGTGGTCCCTCCGGGGATGATGGCGGCAACAGTGCTGAGGTCGAATTTGAAAGCACCTATGAGTCAAGTGTTAAGGACTACTACAAGGATGACCATTCAGTGGACATTGAGAATCATGTCATCCACCCTCCCCCTGTTTTCCACCCTCCACCTGTTCCGGGGTTCCGCAAGAGAGGTGGGGGTGACACCACAGTCATTGGTGGTCCCTCCGGGGATGATGGCGGCAACAGCGCTAGCTTCGAGTTTGATAGCTCATATGCCTCCAGCGTGAAGGATTGGTATAAAGATGATCATTCTGTTGATGTCAAGAATACTATTATCCACCCGCCTCCTATCTTCCACCATCCTCCAGGTGTGGGTTTCCGCAAGAGAGGTGGGGGTGACACCACAGTCATTGGTGGTCCCTCCGGGGATGATGGCGGCAACAGCGCTAGCTTCGAGTTTGACAGCTCATATGCCTCCAGCGTGAAGGATTGGTATAAAGATGATCACTCTGTTGATGTCAAAAACACCGTCATCCATCCTCCCCCTGTCTTCCACCCTCCTCCGGTGCAAGGC

PSRGFHKRGGGDTTVIGGPSGDDGGNSAEVEFESTYESSVKDYYKDDHSVDIENHVIHPPPVFHPPPVPGFRKRGGGDTTVIGGPSGDDGGNSASFEFDSSYASSVKDWYKDDHSVDVKNTIIHPPPIFHHPPGVGFRKRGGGDTTVIGGPSGDDGGNSASFEFDSSYASSVKDWYKDDHSVDVKNTVIHPPPVFHPPPVQG

ii. AF8. 2 repeats

CATCTCGCGGTTTCCATAAGAGAGGTGGGGGTGACACCACAGTCATTGGTGGTCCCTCCGGGGATGATGGCGGCAACAGCGCTAGCTTCGAGTTTGATAGCTCATATGCCTCCAGCGTGAAGGATTGGTATAAAGATGATCATTCTGTTGATGTCAAGAATACTATTATCCACCCGCCTCCTATCTTCCACCATCCTCCAGGTGTGGGTTTCCGCAAGAGAGGTGGGGGTGACACCACAGTCATTGGTGGTCCCTCCGGGGATGATGGCGGCAACAGCGCTAGCTTCGAGTTTGACAGCTCATATGCCTCCAGCGTGAAGGATTGGTATAAAGATGATCACTCTGTTGATGTCAAAAACACCGTCATCCATCCTCCCCCTGTCTTCCACCCTCCTCCGGTGCAAGGCT

PSRGFHKRGGGDTTVIGGPSGDDGGNSASFEFDSSYASSVKDWYKDDHSVDVKNTIIHPPPIFHHPPGVGFRKRGGGDTTVIGGPSGDDGGNSASFEFDSSYASSVKDWYKDDHSVDVKNTVIHPPPVFHPPPVQG

D) Afu6g14090

i. Af293. 40 repeats

GCTGGTGGCTCTAGCTCCGGCTCCGGCTCCGGCTCCGACTCCGGCTCTGGCTCTGGCTCTGGTTCTGGCTCGGGCTCTGGTTCTGGCTCTGGCTCTGGCTCTAGCTCCGGCTCTGGTTCTGGCTCGGGCTCCGGTTCTGGCTCTGGTTCTGGCTCCAACTCCGGCTCTGGCTCTGCCTCTTCCACCGCTACTGGCACCGCTACTGGCACCGCTACTGGCACCGCTACTGGCACCGCTACTGGCACCGCTACT

AGGSSSGSGSGSDSGSGSGSGSGSGSGSGSGSGSSSGSGSGSGSGSGSGSGSNSGSGSASSTATGTATGTATGTATGTATGTAT

ii. AF8. 34 repeats

AGGSSSGSGSGSDSGSGSGSGSGSGSGSGSGSGSSSGSGSGSGSGSGSGSGSNSGSGSASSTATGTATGTAT

iii. AF5. 38 repeats

AGGSSSGSGSGSDSGSGSGSGSGSGSGSGSGSGSSSGSGSGSGSGSGSGSGSNSGSGSASSTATGTATGTATGTATGT