errors "illegal affect", "disconnected", "check_ped_relations" WARNING -> Unconnected entries

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Angela Parody

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Oct 24, 2017, 5:53:45 PM10/24/17

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Hi,

I am trying to convert my plink .bed/.fam/.bim files into EIGENSTRAT format (for subsequent analyses). I encounter this (see below) after the missing Value menu. I have several error messages:

1) first there is this error message (see highlighted in yellow) that says "illegal affect". It looks like it refers to the phenotype (which is quantitative) but I don't know what is the problem. Does anyone knows?

2) Then, see below highlighted in blue. Next menu is about pedigree. I am working with a wild species so I don´t have pedigree information. The .fam file looks like this (I copied the first lines), where first column is family ID or FID (all are 0 because I don't have this information), then the individuals ID or IID, then mother, then father and last are sex and phenotype.

0 NWZ_106801_P03_WH01 0 0 2 85

0 NWZ_106801_P01_WD11 0 0 2 81

0 NWZ_106801_P03_WG01 0 0 1 77

0 NWZ_106801_P02_WG03 0 0 2 80

0 NWZ_106801_P01_WG07 0 0 2 80

0 NWZ_106801_P03_WH05 0 0 2 69

0 NWZ_106801_P03_WG02 0 0 2 80

0 NWZ_106801_P01_WF11 0 0 2 72

0 NWZ_106801_P03_WE06 0 0 2 69

0 NWZ_106801_P03_WE03 0 0 2 81

See also other error messages that comes up (see below highlighted in green). Any idea of what I might be doing wrong and what should I do?

Thanks in advance,

Cheers,

Ángela

==========================================================

Mega2 4.9.2 Missing Value menu:

==========================================================

If it is necessary, specify a different value to indicate that a trait is missing

both for input to Mega2 and/or output from Mega2.

Note: Output entries that are marked with a "#" can not be changed.

0) Done with this menu - please proceed

1) Specify input missing value for Quantitative traits: -9

2) Specify input missing value for Affection status: 0

#) Fixed output missing value for Quantitative traits: *

#) Fixed output missing value for Affection status: *

Select from options 0-2 > 0

Quantitative Input Missing Value -9

Affection Input Missing Value "0"

Quantitative Output Missing Value "*"

Affection Output Missing Value "*"

Input Format: PLINK binary PED format (bed)

Pedigree and map files specified as PLINK format.

omit, penetrance, and frequency files are always in Mega2 format.

Input files will be read in as PLINK or Mega2 format files as appropriate.

Reading PLINK map file for names: myplinkRFsexpheno.bim

Reading map file myplinkRFsexpheno.bim ... (6 columns)

Input Map name: Map, type: average genetic map, units: kosambi Morgans

Input Map name: BP, type: physical map

Found 2 possible maps in the myplinkRFsexpheno.bim file.

Now checking each record in map file myplinkRFsexpheno.bim ...

===== Errors/warnings of type "bad_position":

WARNING: AUTO scaffold03247_107_C_T (map Map, line 1): Missing average position with no sex-specific positions given.

WARNING: AUTO scaffold03247_108_T_C (map Map, line 2): Missing average position with no sex-specific positions given.

WARNING: AUTO scaffold08021_177_G_A (map Map, line 3): Missing average position with no sex-specific positions given.

WARNING: AUTO scaffold12955_202_G_A (map Map, line 4): Missing average position with no sex-specific positions given.

WARNING: AUTO scaffold06360_218_T_C (map Map, line 5): Missing average position with no sex-specific positions given.

WARNING: AUTO scaffold06360_226_A_G (map Map, line 6): Missing average position with no sex-specific positions given.

WARNING: AUTO scaffold06360_234_T_G (map Map, line 7): Missing average position with no sex-specific positions given.

WARNING: AUTO scaffold02723_235_G_C (map Map, line 8): Missing average position with no sex-specific positions given.

WARNING: AUTO scaffold12955_235_C_T (map Map, line 9): Missing average position with no sex-specific positions given.

WARNING: AUTO scaffold03142_264_A_C (map Map, line 10): Missing average position with no sex-specific positions given.

===== Too many "bad_position" records, display is temporarily suspended ..

===== 3412 total records of type "bad_position" are in MEGA2.DB.ERR

Done reading map file: myplinkRFsexpheno.bim

===== Errors/warnings of type "unmapped_errors":

WARNING: Locus scaffold03247_107_C_T chromosome is unavailable.

WARNING: Locus scaffold03247_108_T_C chromosome is unavailable.

WARNING: Locus scaffold08021_177_G_A chromosome is unavailable.

WARNING: Locus scaffold12955_202_G_A chromosome is unavailable.

WARNING: Locus scaffold06360_218_T_C chromosome is unavailable.

WARNING: Locus scaffold06360_226_A_G chromosome is unavailable.

WARNING: Locus scaffold06360_234_T_G chromosome is unavailable.

WARNING: Locus scaffold02723_235_G_C chromosome is unavailable.

WARNING: Locus scaffold12955_235_C_T chromosome is unavailable.

WARNING: Locus scaffold03142_264_A_C chromosome is unavailable.

===== Too many "unmapped_errors" records, display is temporarily suspended ..

===== 3412 total records of type "unmapped_errors" are in MEGA2.DB.ERR

===========================================================

Total number of loci = 3413

1 trait locus

1 Affection status locus:

default

3412 Marker loci

3412 Unmapped loci

Number of loci found per chromosome (chromosome:number)

U:3412

===========================================================

WARNING: No frequency file provided.

WARNING: Allele frequencies for these will be estimated from data.

Trait 'default' will be assigned the default penetrance: (0.0500 0.9000 0.9000)

Checking PLINK Binary format file: myplinkRFsexpheno.bed

Detected PLINK .bed file v1.00 SNP-major mode.

===== Errors/warnings of type "illegal_affect":

ERROR: Line 1, column 5: Illegal affection status 85, setting to unknown.

ERROR: Line 2, column 5: Illegal affection status 81, setting to unknown.

ERROR: Line 3, column 5: Illegal affection status 77, setting to unknown.

ERROR: Line 4, column 5: Illegal affection status 80, setting to unknown.

ERROR: Line 5, column 5: Illegal affection status 80, setting to unknown.

ERROR: Line 6, column 5: Illegal affection status 69, setting to unknown.

ERROR: Line 7, column 5: Illegal affection status 80, setting to unknown.

ERROR: Line 8, column 5: Illegal affection status 72, setting to unknown.

ERROR: Line 9, column 5: Illegal affection status 69, setting to unknown.

ERROR: Line 10, column 5: Illegal affection status 81, setting to unknown.

===== Too many "illegal_affect" records, display is temporarily suspended ..

ERROR: Found 1 records with invalid sex value.

3412 (of 3412) markers to be included from myplinkRFsexpheno.bim

Reading pedigree information from myplinkRFsexpheno.fam

265 individuals read from myplinkRFsexpheno.fam

265 individuals with nonmissing phenotypes

0 cases, 0 controls, 0 missing

128 males, 136 females, 1 of unspecified sex

0 founders, 265 non-founders found

===========================================================

Input pedigree data contains:

Input pedigree file is in PLINK-fam format.

Marker Genotypes

Fully Half

Pedigrees People Males Females Typed Typed Total

TOTAL 1 265 128 136 901712 0 904180

Typed 1 264 128 136

Untyped 0 1 0 0

===========================================================

Pedigree exclusion option : Include all pedigrees whether typed or not.

Select individuals to compute allele frequencies

for recoded marker loci:

==========================================================

0) Done with this menu, please proceed.

1) Genotyped founders only

2) Genotyped founders + a randomly chosen genotyped person

from pedigrees without genotyped founders.

3) Genotyped founders + genotyped individuals with unique alleles

*4) All genotyped individuals

5) Count half-typed individuals' alleles. [no ]

Select from options 0 - 4 or 5 to toggle > 0

Count option: all alleles

Count half-typed individuals' alleles : no

===========================================================

Recoding pedigree genotypes ...

===========================================================

Pedigree data summary after recoding:

Input pedigree file is in PLINK-fam format.

Marker Genotypes

Fully Half

Pedigrees People Males Females Typed Typed Total

TOTAL 1 265 128 136 901712 0 904180

Typed 1 264 128 136

Untyped 0 1 0 0

===========================================================

===== Errors/warnings of type "disconnected":

ERROR: Pedigree 0 has disconnected individuals: 0_NWZ_106801_P03_WH01

ERROR: Pedigree 0 has disconnected individuals: 0_NWZ_106801_P01_WD11

ERROR: Pedigree 0 has disconnected individuals: 0_NWZ_106801_P03_WG01

ERROR: Pedigree 0 has disconnected individuals: 0_NWZ_106801_P02_WG03

ERROR: Pedigree 0 has disconnected individuals: 0_NWZ_106801_P01_WG07

ERROR: Pedigree 0 has disconnected individuals: 0_NWZ_106801_P03_WH05

ERROR: Pedigree 0 has disconnected individuals: 0_NWZ_106801_P03_WG02

ERROR: Pedigree 0 has disconnected individuals: 0_NWZ_106801_P01_WF11

ERROR: Pedigree 0 has disconnected individuals: 0_NWZ_106801_P03_WE06

ERROR: Pedigree 0 has disconnected individuals: 0_NWZ_106801_P03_WE03

===== Too many "disconnected" records, display is temporarily suspended ..

===== 265 total records of type "disconnected" are in MEGA2.DB.ERR

Created linkage ped tree

NOTE: Only one genetic map is available for use, so it will be used.

NOTE: Only one physical map is available for use, so it will be used.

Done checking locus integrity.

Checking pedigree integrity...

===== Errors/warnings of type "check_ped_relations":

ERROR: Ped 0: Found 265 disconnected sub-pedigrees:

ERROR: Sub-pedigree 1 including (but not limited to) individuals

0_NWZ_106801_P03_WH01

ERROR: Sub-pedigree 2 including (but not limited to) individuals

0_NWZ_106801_P01_WD11

ERROR: Sub-pedigree 3 including (but not limited to) individuals

0_NWZ_106801_P03_WG01

ERROR: Sub-pedigree 4 including (but not limited to) individuals

0_NWZ_106801_P02_WG03

ERROR: Sub-pedigree 5 including (but not limited to) individuals

0_NWZ_106801_P01_WG07

ERROR: Sub-pedigree 6 including (but not limited to) individuals

0_NWZ_106801_P03_WH05

ERROR: Sub-pedigree 7 including (but not limited to) individuals

0_NWZ_106801_P03_WG02

ERROR: Sub-pedigree 8 including (but not limited to) individuals

0_NWZ_106801_P01_WF11

ERROR: Sub-pedigree 9 including (but not limited to) individuals

0_NWZ_106801_P03_WE06

===== Too many "check_ped_relations" records, display is temporarily suspended ..

===== 266 total records of type "check_ped_relations" are in MEGA2.DB.ERR

Done checking pedigree integrity.

==========================================================

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

WARNING: Thus far found these problems/errors in input data (see MEGA2.ERR for details):

WARNING: -> Unconnected entries

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

==========================================================

Specify whether to reset Mendelianly inconsistent loci to missing:

==========================================================

0) Done with this menu - please proceed

1) Set all genotypes to unknown within entire pedigrees

at each Mendelianly-inconsistent locus? [no ]

If "no" is indicated, Mendelianly-inconsistent loci

will not be looked for.

2) EXIT Mega2.

Select from options 0-2, 1 toggle >

Dan Weeks

unread,

Oct 25, 2017, 11:52:29 AM10/25/17

to Mega2

Dear Angela,

To treat each of the individuals as unrelated, then you need to give each of them a unique family ID (FID). Currently, since you gave them all the same family ID of "0", Mega2 thinks they are all members of a single pedigree, but then complains because you have not specified the pedigree structure via the mother and father ID columns.

When reading in PLINK files, Mega2 defaults to assuming that the trait is an affection status trait. Since your trait is quantitative instead, then you have to use Option 2 of the Mega2 file input menu to tell Mega2 that via the "--quantitative" flag, as described in Section 9.2.1 "PLINK PED input format" of the Mega2 documentation:

To read the example PED file correctly while naming the quantitative trait “Q1”, we use the Mega2 PLINK parameters:

--trait Q1 --quantitative

by setting them in Option 2 “Enter PLINK parameters” of the Mega2 file input menu.

I hope that this information will help you get Mega2 working on your data.

Sincerely,

Dan

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