Gene Finding on D. biarmipes contig 16

Jolene Rearick - June 18, 2013

Summary

D. biarmipes contig 16 encodes two genes: the D. biarmipes orthologs of the RhoGAP102A (Gene B) and dpr7 (Gene A) D. melanogaster genes.

BLASTX analysis

I used the file contig16.fasta from the src folder in the GEP project file for D. biarmipes contig as a query sequence in a BLASTX search of Non-redundant protein sequences (nr) restricted to Drosophila melanogaster. All BLAST parameters were left at the default settings.

Graphic summary. The graphic summary is shown below.

The BLASTX search suggests there are two genes on the contig with protein sequence similarity to the D. melanogaster protein set.

The left gene (Gene A) has many good matches to D. melanogaster proteins. The two best matches are different isoforms of the same gene. Other matches are still good and appear to be copies of the gene.

The right gene (Gene B) has four good matches. These appear to be isoforms derived from alternative splicing. There are many poor-scoring hits that cover only a small portion of the top hit.

Descriptions. All descriptions with E values smaller than e-10 are shown below.

Alignments

Gene B. The first four alignments in the list of descriptions all align to a region of the contig corresponding to Gene B in the graphic summary. The coordinates of the top hits are shown in the table below.

Reordering these in order of the segments of NP_001033802.2 gives the results shown below.

Summary of Gene B: Gene B appears to be the D. biarmipes ortholog of the D. melanogaster gene RhoGAP102A. The gene is on the plus strand (all matching reading frames are 1, 2, or 3). The entire gene appears to be on the contig. A view of D. melanogaster RhoGAP102A from FlyBase GBrowse is shown below.

The D. melanogaster RhoGAP102A gene has four isoforms derived from alternative splicing in the 3' of the gene.

To confirm this, I used the Gene Record Finder at GEP. This returns the following protein sequences for the exons of D. melanogaster RhoGAP102A:

>RhoGAP102A:1_1653_0
MEFPKENEESVNCLRSPMAAVKKRSGPLDIALDEANDVSKIWSLPLGNAS
SMRKPMIFASEHSEGVASPNKVRRRSTKDKEKKRERWLLTRKTWRYMTDA
GRKLIPDGYQTGSGNHDLIEDQFQRVCLSEPSFILWSRRTSYPGAFSCSK
RRLKPLLRHASASRRKQTVDATKDYHIADRVIELLQTYLKLRDAYKTTTL
LGTKTVRPDQTSSPSAQRPNFKNNGDNHQVISSGTSIQTELLSLLKLLSN
CPLFTHEGIQLKFGDAPLSILEDKVLLKKIYSALKKQQLHRTLHSKDPYQ
ANFSTSLSCLIKNGKELSRGNTDSNRSNKLLHDNFFCLMKCDTKPIIPSP
KTEFFLDLNKVPQNFELKNKLNSNGQSVERIQKTCGTQTNFIQLSELKTL
AEQYNLMVTNCDNTLKLLEEPSKQDCSKSSHLIFRRSSLDEDISQSVSDT
IKRYLNMARKKSMQDSDSNRFKSINYDKNLRNIKAKGVLNPPGISAGLHK
AVQTLNAWPLIALDFIRGNESSLNLKNAHLEWLRLEDERSHMQLERDKNP
IELRSKVNPLQIASPANTSSYSKCTSAPTSPTSHSKLEKAIRTSSGLLSS
SSQFISNILHGHNNAGNQFNTLTDKLPDC

>RhoGAP102A:2_1653_2
QSHATASMQKSKSLSNVGQFVAKRMWRSRSKSQNKRSYLKSSIPTLKWYP
S

>RhoGAP102A:3_1653_0
EHFLWISESGESFQIVETNLTRLSKIESDIVKNFALEKIQELNIGNIE

>RhoGAP102A:4_1653_2
LKISPQKRRCVPKKKSLTTSFFDNGKKDDQ

>RhoGAP102A:5_1653_2
PRSVLFGTSLECCLERDSKRNVTMEDRSKYSLLSMFRGSGSTPGSVLKLN
DN

>RhoGAP102A:6_1653_0
VRSCESLPSKSLEYGSTELSAYVRGSFSNISKPLASLSQSENEDTELNFV
KAYQEHSQLMVPIFVNNCIDYLEDNGLQQVGLFRVSTSKKRVKQ

>RhoGAP102A:7_1653_0
LREEFDKDIYFGISVDTCPHDVATLLKEFLRDLPEPLLCNTLYLTFLKTQ

>RhoGAP102A:9_1653_2
IRNRRLQLEAISHLIRLLPIPHRDTLYVLLVFLAKVAAHSDDIWSTEGCC
LTLGNKMDSYNLATVFAPNILRSTHLTFSRDKEQENMIDAINVVRFVKQN
TYLLFMPLIRPNLPSR*

>RhoGAP102A:8_1653_2
IRNRRLQLEAISHLIRLLPIPHRDTLYVLLVFLAKVAAHSDDIWSTEGCC
LTLGNKMDSYNLATVFAPNILRSTHLTFSRDKEQENMIDAINVV

>RhoGAP102A:10_1653_1
RRGSKGTEEPQPKRKWISRPGPDAQQKSPVMLLPHASYKKYSDILSLRLR
QELKLERTNLERRAFIHINNKECNGLGSRPADTR*

>RhoGAP102A:11_1653_1
TMINHYEEIFNISAELLNVIYTQALEACPEKLYELISTKVYGTE

>RhoGAP102A:12_1653_1
TQQQIDDPQPGSLSDVLLEPPIHGK

>RhoGAP102A:13_1653_1
YENINDFNNINFKRCQDKRRDHSDPDKKKNNNENLEIITASLKISVAEQS
HISLKEPIKEQ

>RhoGAP102A:14_1653_1
CQDKRRDHSDPDKKKNNNENLEIITASLKISVAEQSHISLKEPIKEQ

>RhoGAP102A:15_1653_2
PITSYKQFSRSTLPTSISDVGVNTLRTDGAKLENKMKILTSNINKQDIPI
KTTYKRQNLISSSRRISQEP*

These sequences were used as subject sequences in a bl2seq search with BLASTX using contig16.fasta as the query sequence.

The RhoGAP102A gene of D. melanogaster is approximately 10 kb, all of which appears to be on the contig. The first exon was a good match (E = 1E-179) to the 23,501 - 25,117 portion of the contig in frame +2. An additional match was also found in this region, overlapping the first exon in a different reading frame (+3, E = 6E-17, contig16 23,280 - 23,516), but was rejected due to being a poorer match on the same strand in a different frame. In addition, the first exon of RhoGAP102A in D. melanogaster is approximately 2 kb, as is the better match to the first exon. The worse match is only 0.3 kb long. The second exon has only one good match (E = 2E-14) from contig 26,883 - 27,035. The third exon has one good match from contig 27,095 - 27,238 with E = 7e-18. Exon four of RhoGAP102A matches 29,060 - 29,149(E = 7E-10).

Alignments

Gene A. The fifth and sixth alignments in the list of descriptions align to a region of the contig corresponding to Gene A in the graphic summary. The coordinates of the top hits are shown in the table below.

Reordering these in order of the segments of NP_001096850.2 gives the results shown below.

Summary of Gene A: Gene A appears to be the D. biarmipes ortholog of the D. melanogaster gene dpr7. The gene is on the minus strand (all matching reading frames are -1, -2, or -3). The entire gene appears to be on the contig. A view of D. melanogaster dpr7 from FlyBase GBrowse is shown below.

The D. melanogaster dpr7 gene has two isoforms derived from alternative splicing in the last two exons.

To confirm this, I used the Gene Record Finder at GEP. This returns the following protein sequences for the exons of D. melanogaster dpr7:

>dpr7:9_1642_0
MSRGKHATFLNNSPIKIKPFIILVLNLNIWCQYISATSYLSLN

>dpr7:8_1653_0
MKTYNAFLNTKTKIIHV

>dpr7:7_1642_2
LTNLERPFFDDISPRNVSAVVDEIAILRCRVKNKGNRT

>dpr7:6_1642_0
VSWMRKRDLHILTTNIYTYTGDQRFSVIHPPGSEDWDLKIDYAQPRDSGV
YECQVNTEPKINLAICLQVI

>dpr7:5_1642_2
DNDFQDLKTKKRFYDTK

>dpr7:4_1642_2
ARAKILGSTEIHVKRDSTIALACSVNIHAPSVI

>dpr7:3_1642_1
YHGSSVVDFDSLRGGISLETEKTDVGTTSRLMLTRASLRDSGNYTCVPNG
AIPASVRVHVLT

>dpr7:2_1642_2
EQPAAMQTSSAIRIRAFTAMITIISTKVLLYISSLMEHMYLRER*

These sequences were used as subject sequences in a bl2seq search with BLASTX using contig16.fasta as the query sequence.

????????????????Needs fixing for unreported transcript??????? The dpr7 gene of D. melanogaster is approximately 6 kb, all of which appears to be on the contig. However, the first two exons from the left give no significant match (respectively exons 1 and 2, E = 0.84, and 0.46). The fifth exon gives a significant match (E = 8E-19) to 17,328 - 17,215 on the contig. The fourth exon also gives a significant match located at contig 12,808 - 12,602 (7E-42). The third exon does not give a significant match (7e-05). The second exon gives a significant match (1E-36) from contig 11,701 - 11,516. The first exon of dpr7 gives a significant match to the contig from 11,458 - 11,324, (2E-19).

BLAST analysis by GEP

The project folder from GEP contains the following analysis (analysis/BLASTresults/contig16_dmel_translation.blx):

                                                                     Smallest
                                                                       Sum
                                                              High  Probability
Sequences producing High-scoring Segment Pairs:              Score  P(N)      N

RhoGAP102A-PD FlyBase:FBpp0300897                             1669  0.       13
RhoGAP102A-PF FlyBase:FBpp0306510                             1669  0.       13
RhoGAP102A-PC FlyBase:FBpp0289000                             1669  0.        9
RhoGAP102A-PE FlyBase:FBpp0289001                             1669  0.        9
dpr7-PF FlyBase:FBpp0306511                                    364  9.3e-163  7
dpr7-PG FlyBase:FBpp0306512                                    364  3.3e-149  6
dpr8-PA FlyBase:FBpp0073730                                    234  1.8e-50   5
dpr4-PB FlyBase:FBpp0289718                                    244  6.4e-49   5
dpr6-PA FlyBase:FBpp0076082                                    211  6.8e-49   4
dpr6-PC FlyBase:FBpp0303926                                    211  3.4e-48   4
dpr6-PE FlyBase:FBpp0304912                                    211  3.5e-48   4
dpr6-PD FlyBase:FBpp0304911                                    211  9.9e-48   4
CG42596-PC FlyBase:FBpp0306958                                 200  5.7e-42   5
dpr9-PA FlyBase:FBpp0088403                                    246  6.7e-42   4
dpr-PA FlyBase:FBpp0099910                                     252  2.0e-40   4
dpr4-PC FlyBase:FBpp0289719                                    244  2.4e-40   4
dpr2-PD FlyBase:FBpp0297253                                    263  4.3e-39   5
dpr2-PE FlyBase:FBpp0297254                                    263  8.8e-39   5
dpr10-PA FlyBase:FBpp0076037                                   206  4.7e-35   4
dpr10-PB FlyBase:FBpp0076038                                   206  4.7e-35   4
dpr10-PC FlyBase:FBpp0076039                                   206  4.7e-35   4
dpr5-PB FlyBase:FBpp0081871                                    227  7.6e-35   5
dpr5-PA FlyBase:FBpp0081870                                    227  2.0e-34   5
dpr10-PD FlyBase:FBpp0303942                                   206  7.6e-32   3
dpr3-PB FlyBase:FBpp0289895                                    208  2.8e-25   5
dpr3-PC FlyBase:FBpp0306835                                    208  2.8e-25   5
dpr3-PD FlyBase:FBpp0306836                                    208  2.8e-25   5
dpr11-PB FlyBase:FBpp0081202                                   180  1.1e-24   5
dpr11-PC FlyBase:FBpp0307559                                   180  1.1e-24   5
dpr12-PD FlyBase:FBpp0307251                                   188  2.3e-24   5
dpr12-PC FlyBase:FBpp0289780                                   188  2.5e-24   5
dpr12-PE FlyBase:FBpp0307252                                   188  2.2e-23   5
dpr13-PB FlyBase:FBpp0289452                                   199  1.5e-22   5
dpr14-PA FlyBase:FBpp0071086                                   140  5.7e-21   4
dpr14-PB FlyBase:FBpp0298271                                   140  5.7e-21   4
dpr15-PA FlyBase:FBpp0082022                                   192  1.8e-18   5
dpr17-PB FlyBase:FBpp0082017                                   161  6.2e-17   2
dpr17-PC FlyBase:FBpp0289403                                   161  6.2e-17   2
dpr17-PA FlyBase:FBpp0082016                                   161  1.2e-16   2
dpr19-PA FlyBase:FBpp0079594                                   134  9.2e-15   4
dpr19-PB FlyBase:FBpp0304500                                   134  9.2e-15   4
dpr19-PC FlyBase:FBpp0304501                                   134  9.2e-15   4
dpr20-PA FlyBase:FBpp0072491                                   106  1.4e-12   2

I combined isoforms into single genes, then identified the segment of the contig aligning to each sequence, as summarized in the table below. A representative protein sequence (NP_xxxxx) from D. melanogaster was chosen for each gene (only one isoform for genes with multiple isoforms) and used as the subject sequence for a bl2seq search with BLASTX using contig16.fasta as the query sequence. All aligning segments were combined to give a coordinate range on the contig. The E value for the highest scoring segment is shown.

FlyBase BLAST

I used the link to FlyBase BLAST from the Tools page of the course website.

I set the Database to Annotated proteins (AA), the Program to BLASTX, and uploaded the fosmid sequence. I restricted the species to D. melanogaster and clicked BLAST.

The graphic output is shown below. Notice that each hit is labeled, unlike the results at NCBI BLAST.

A summary table is also shown below.

GENSCAN analysis

The GENSCAN results from analysis/Genefinder/Genscan in the project folder predict four proteins on the contig:

>contig16|GENSCAN_predicted_peptide_1|210_aa
MPRGNNATYSDNSSIKIKPFIILILNFNICLQQINASSFLNFNDLTSSDKPYFDDISPRN
VSAVVDEIAILRCRVKNKGNRTVSWMRKRDLHILTTNIYTYTGDQRFSVIHPPSSEDWDL
KIDYAQPRDSGVYECQVNTEPKINLPIVLEITDFDSLRGGISLETEKTEIGTTSRLMLTR
ASLRDSGNYTCVPNGAIPASVRVHVLTGKH

>contig16|GENSCAN_predicted_peptide_2|929_aa
MTDAGRKLIPDSYPAGSDNFDLLEEHFQRVCLSEPSFILWNRRTSYPGAINSSRRRLKQL
SRHACSSHKENSIETKNSYYNADRTIELLQTFLKLRDAYKTTTLLGTKTVRPDQTSSPSA
QRPIFGKKSEKDQTMSSDMTLQKELICRLKLLSSCINFAQVGIKLEISDLSETILEDKVL
LKKIYSALKKQQLHRTLHSKEPNKKSVNRSSSLSSLKIGEHESMSSKTSEDQKLKNQNFQ
CVKICDTSSKNSINLDLNCVIKSIEFPNKCLLSDQKIERSVKTCGTQTSFIQLSELKSLA
EQYKCMVQNCDNNLQVFQEFDKQDGLTSSRSTCRKSSIDEDISQSVSDTIKRYLKMARKK
SVQGSDSNRFKSVNYDQNLKNIKAKGEINPPALNDGLNKAVQTLDAWPVIALDFIKGNES
SIYLQNAHLEWIRSEDEREQKQLEWNKKQKQIDKEEHTPHEINRGNASHYSTCTSAPTSP
TSHSKLEKAIRTSSGLLSSSSQFISSILHGHSSAGSQYSNLGNDSVNMQKSKSLSNVGQF
VSKKIWGSRFKSQSKRNFSKGLKDLPSVKWHPSDNCIWISEDGERFQIVDTLLIRLSKRE
TDLVKDFALEKIEELNIGNIDDLKKTSKKRRIAPKKKSLTTSFFDIGKKDDQNERVALFG
TSLECCLARDRKGSANIEDRSEHYVFRKSGSNPGSVMKLNDNVRSCESLPSKSLEFGYMD
SSDCSSGSFNTIPKPAASLTQFEIGDTEPSFYKTYQDQLILMVPMFIINCIEYLEENGLQ
KVGLFRVSTSKKRVKQPFCNIRGEGCVRTDRRVVSYGNHTPIYIKLYQPNSFGKQKGWHT
SLYVDFRKLNSMVDEQRYSRFIHECYSADTVESCLQKMALVLNTARAFGLQNKCNFLQTQ
ILFLVRNIEKGNLWPGEDKTAAVSKFSNT

>contig16|GENSCAN_predicted_peptide_3|72_aa
MINHYEEIFKISAELLDVIYTRVMEACPEQLYELISMKLNGYEWNLNQLDDPQPSSLGDV
MFEPAVQEKRFV

>contig16|GENSCAN_predicted_peptide_4|51_aa
PSKEDLTAVSHKPPQHNLRPPARLSREDTGMARKPSNPCYTNLILEGVISE




Query
Top hit
E
Coverage
Max identity



Accession
Gene



GENSCAN_predicted_peptide_1
NP_001096850.2
dpr7

100%
79%



GENSCAN_predicted_peptide_2
NP_001033802.2
RhoGAP102A
0.0
85%
63%



GENSCAN_predicted_peptide_3
NP_001245416.1
RhoGAP102A
3e-28
98%
68%



GENSCAN_predicted_peptide_4
NP_649295.1
CG9389
1.8
88%
24%

Because no significant matches were found with peptide 4, I repeated the BLASTP search of nr with the species restriction turned off. Again, no significant similarities were found.

Summary of GENSCAN analysis: GENSCAN identified three genes found by the BLASTX search (Hem, Aats-ile, and Ten-m). It made two additional predictions that are invalid.

Here is a view of contig16 in the UCSC Genome Browser at GEP.

BLASTX Alignment of D. melanogaster proteins. The BLASTX track at the top of the image shows alignments to two overlapping regions, as was seen in the prior BLASTX analaysis. The left D. biarmipes gene, dpr7, aligns to the protein product of the D. melanogaster dpr7 gene and many homologs of this gene. The right D. biarmipes gene, RhoGAP102A, aligns to the D. melanogaster RhoGAP102A gene. These are the same results seen when the contig is used as a query sequence in a BLASTX search of D. melanogaster proteins. There are two genes on the contig: the D. biarmipes orthologs of dpr7 and RhoGAP102A.

GENSCAN predictions. Starting at the left of the contig, the first three GENSCAN predictions align to the D. biarmipes orthologs of dpr7 and RhoGAP102A. The fourth GENSCAN prediction does not align to sequences predicted by BLASTX analysis to encode proteins. The first GENSCAN prediction is congruent with predictions from other gene-finding programs, while the second and third predictions split the RhoGAP102A gene.

modENCODE RNA-Seq. Transcripts aligning to dpr7 and RhoGAP102A are seen. In addition there are transcripts in the 5' end of the contig at approximately 1 kb. These transcripts are a portion of the dpr7 gene that was not detected in any other gene-finding analysis.

Conservation. ??????????????????????Need to fix this part??????????????????The exons of Hem, Aats-ile, and Ten-m are clearly conserved. The intergenic regions between Hem and Aats-ile, and between Aats-ile and Ten-m are not conserved. There is considerable sequence conservation upstream of the rightmost (5') exon in Ten-m; this region is known to be a Ten-m intron, separating the third exon of Ten-m from the second exon, which is not on the fosmid.