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Table 1 methBLAST results of a panel of assays from methPrimerDB (1–12) and literature (13–14).

From: methBLAST and methPrimerDB: web-tools for PCR based methylation analysis

    

Best hit when both primers align on correct target

First misalignment

#

ID

Assay type

Gene symbol

Sequence identifier

Score (bits)

E value

Sequence identifier

Score (bits)

E value

1

14

BiPS

VHL

gb|L15409.1| [BISUL_METH_RC]

40.1 (20)

0.32

dbj|D64176.1| [BISUL_METH_RC]

32.2 (16)

79

2

236

BiPS

NKX3-1

gb|U91540.1| [BISUL_UNMETH_FW]

46.1 (23)

0.007

dbj|AP005361.2| [BISUL_UNMETH_RC]

44.1 (22)

0.026

3

26

Ms-SNuPE

RARB

emb|X56849.1| [BISUL_UNMETH_FW]

44.8 (22)

0.015

dbj|AP001217.3| [BISUL_UNMETH_RC]

40.8 (20)

0.25

4

28

Ms-SNuPE

CDKN2A

dbj|AB060808.1| [BISUL_UNMETH_RC]

46.1 (23)

0.007

emb|AL590456.5| [BISUL_UNMETH_RC]

40.1 (20)

0.41

5

13

COBRA

ESR1

gb|M69297.1| [BISUL_UNMETH_RC]

46.1 (23)

0.006

gb|AE014307.1| [BISUL_UNMETH_RC]

40.1 (20)

0.40

6

16

COBRA

FLT1

emb|AL139005.12| [BISUL_UNMETH_RC]

40.8 (20)

0.22

emb|AL731546.4| [BISUL_UNMETH_FW]

38.8 (19)

0.91

7

24

BiSeq

CDKN2C

gb|AY094608.1| [BISUL_UNMETH_FW]

50.1 (25)

4e-04

gb|AC027334.5| [BISUL_UNMETH_FW]

40.1 (20)

0.43

8

83

BiSeq

SERPINB5

gb|AC036176.8| [BISUL_UNMETH_RC]

60.0 (30)

7e-07

emb|AL133413.5| [BISUL_UNMETH_FW]

52.0 (26)

2e-04

9

1

MSP-unmeth

VHL

gb|AC018808.5| [BISUL_UNMETH_RC]

48.1 (24)

0.002

emb|AL513423.3| [BISUL_UNMETH_RC]

44.1 (22)

0.025

10

1

MSP-meth

VHL

gb|L15409.1| [BISUL_METH_FW]

44.1 (22)

0.021

gb|AC079148.9| [BISUL_METH_FW]

36.2 (18)

5.1

11

17

MSP-unmeth

CDKN2A

dbj|AB060808.1| [BISUL_UNMETH_RC]

48.1 (24)

0.002

emb|AL137127.7| [BISUL_UNMETH_RC]

42.1 (21)

0.11

12

17

MSP-meth

CDKN2A

gb|AF044170.1| [BISUL_METH_FW]

48.1 (24)

0.002

emb|AL137077.31| [BISUL_METH_RC]

36.2 (18)

6.4

13

Ref [15]

MSP-unmeth

CDKN2A

dbj|AP001626.1| [BISUL_UNMETH_RC]

38.2 (19)

1.7

emb|AL161774.49| [BISUL_UNMETH_FW]

44.1 (22)

0.028

14

Ref [15]

MSP-meth

CDKN2A

Na

Na

Na

gb|AC092171.4| [BISUL_METH_FW]

38.2 (19)

1.7

  1. This list summarizes the output of 14 individual methBLAST searches. We randomly selected two assays from the five assay types available in methPrimerDB and listed the sequence identifier, the score and E value of the best hit when both the forward and reverse primer align correctly on the desired target and of the first alignment on an undesired target sequence. The MSP assays have two primer pairs resulting in two methBLAST outputs (9–14). All misalignment hits are the result from partial alignment of one of the primers of an assay. This does not result in aspecific amplification. The alignments of the primers from the assays submitted in methPrimerDB are correct and do not demonstrate any potentially amplifiable misalignments. Alignment results #13 and #14 were generated with primers published in literature [15] that are almost identical to the primers from methPrimerDB ID 17 for which the results are numbered as #11 and #12. Result #13 shows correct but incomplete alignment compared to result #12 and attempt #14 does not lead to any correct alignment.
  2. Abbreviations: ID: methPrimerDB ID; BiPS: PCR-single-strand conformation polymorphism analysis; Ms-SNuPE: methylation-sensitive single-nucleotide primer extension; COBRA: combined bisulphite restriction analysis; BiSeq: Bisulphite Sequencing; MSP-unmeth: methylation-specific PCR for unmethylated target detection; MSP-meth: methylation-specific PCR for methylated target detection
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BMC Bioinformatics

ISSN: 1471-2105

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