Gene Finding Methods

Outline

• Overview
• Gene Structure Prediction
• Gene Naming
• Name Counts
• Gene Numbering
• Overview of Query Genes
• Overview of Reference genes
• Splice Analysis
• Possible Problems

Overview

This document explains how automated gene calls were produced for each of the Fusaria genomes. The annotation of the final gene structures were created using a combination of ESTs, predicted ORFs, ab-initio gene predictions and mapped ORFs from either Fusarium graminearium (in the case of FV3) and from Fusarium verticillioides (in the case of FO2). This automated gene calling process is described in Gene Structure Prediction. Genes were assigned names using our automated gene naming system. This process is described in Gene Naming.

Gene Structure Prediction

Gene structures were predicted using a combination of manual annotation, FGENESH, GENEID and EST-based genes called FindORFs. FGENESH is a commercial gene prediction program sold by Softberry, while GENEID, by Enrique Blanco and Roderic Guigo, is available under the GPL.

In FV3, the FGeneSH and GENEID gene sets contained 13,806 and 15,408 gene predictions respectively. Where multiple predictions overlap each other and EST evidence, we choose the one most in accord with the EST splice sites. If we had sufficient EST coverage to build complete ORFs, we replaced any overlapping predicted gene model with the ORF predicted purely from ESTs.

FindORFs is a gene finding program developed at Broad. The 3,856 findOrfs genes were built as follows. First, ESTs are aligned to the genome and grouped into loci consisting of overlapping ESTs. Then, each locus is examined for compatible splicing. If two ESTs in the same locus have identical splice sites where they overlap, they are considered fragments of a larger transcript. Putative transcripts are incrementally built out by adding additional ESTs to either end.

Each putative transcript is built from one or more ESTs, but may not represent the full biological transcript if the EST coverage is incomplete. We search each putative transcript for ORFs beginning with ATG and ending with a stop codon, with no frameshifts. If a putative transcript contains an ORF longer than 180 nt that covers 1/3 or more of its spliced length, we considered it a valid gene prediction. The full set of these predictions is in the group labeled 'findOrfs'. Further, we select only a subset of putative full-length gene models from these EST-based findORF transcripts that are contained totally within the best ab initio gene prediction. An independent blast-based analysis was used also for validating the reading frame by comparing them to the best hit known proteins in the NR database.

Targeted manual annotation was carried out in loci where gene predictions clashed with EST evidence or where EST and BlastX suggested a gene locus but gene predictions were absent. In total, 1016 manual annotations were carried out. At all loci with no manually annotated gene models but containing EST-based findOrfs, we picked the full-length findOrfs over any ab-initio gene predictions.

In FG3, the FGeneSh and GeneID genesets contained 11,782 and 12,670 gene predictions respectively. The final gene set from FV3 were mapped onto the FG3 genome with 6,994 transcripts successfully transferring. MIPS Gene data from FG1 was mapped onto FG3 also with 13,909 transcripts successfully mapping. FindORFs, as described above, was run on this set and produced 2,431 genes. Targeted manual annotation was carried out in loci where gene predictions clashed with EST evidence or where EST and BlastX suggested a gene locus but gene predictions were absent. In total, 120 manual annotations were carried out. At all loci with no manually annotated gene models but containing ESTs, mapped genes from the MIPS FG1 data and the FV3 gene transfers, we picked ESTs and full-length mapped genes over any ab-initio gene predictions.

In FO2, the FGeneSh and GeneID genesets contained 18,482 and 21,117 gene predictions respectively. The final gene set from FV3 were mapped onto the FG3 genome with 12,840 transcripts successfully transferring. FindORFs, as described above, was run on this set and produced 4,204 genes. Targeted manual annotation was carried out in loci where gene predictions clashed with EST evidence or where EST and BlastX suggested a gene locus but gene predictions were absent. In total, 94 manual annotations were carried out. At all loci with no manually annotated gene models but containing ESTs and mapped genes from the FV3 gene transfers, we picked ESTs and full-length mapped genes over any ab-initio gene predictions.

Gene Naming

Genes are assigned names very conservatively. As this is a purely automated gene prediction process, we do not want to propagate misinformation by transferring unverified functional names for genes in one species to predicted genes in another species.

We hope to improve the gene naming process in the future based on Gene Ontology categories.

There are currently 5 types of gene name that fall into 3 categories:

1. NAME, or hypothetical protein similar to NAME, or conserved hypothetical protein
2. Assigned to gene predictions where there is excellent homology to a known NR protein. The criteria for this category are:
   • At least one BLASTP hit to a known NR protein (complexity filtering off, -F F, expect = 1e-10),
   • A minimum of 50% identity and 70% coverage of both the query and subject sequence.
3. The name will follow one of these three formats:
   • conserved hypothetical protein if the homologous protein NAME contains a word indicating the name has not been verified: {fragment, homolog, hypothetical, like, predicted, probable, putative, related, similar, synthetic, unknown, unnamed}, otherwise
   • NAME if the homologous protein is from the curated Swiss-Prot gene set, otherwise:
   • hypothetical protein similar to NAME Where there is more than one suitable name for a BLAST hit, we prefer Swiss-Prot names to non-Swiss-Prot names. If there are multiple distinct BLAST hits, we choose the one with the highest average identity to the amount of overlap to the target gene. In all cases we take the NR protein name and filter out the species name, GIs, parenthetical comments, extra white space, etc.
   • Hypothetical protein Assigned to gene predictions that show significant BLASTP homology to a protein in NCBI's protein set NR. The criteria for this category are:
     • BLASTP hit to NR (complexity filtering off, -F F, expect = 1e-10)
   • Predicted protein Assigned to gene predictions that do not show significant BLASTP homology to any proteins in NCBI's non-redundant set of proteins (NR) at the time that the complete BLASTP analysis was performed on the gene set.

Name Counts

Name Counts (FO2)

3814 transcript(s) had non-generic names.

"conserved hypothetical protein"	9467
"hypothetical protein"	527
"predicted protein"	3927
hypothetical protein similar to...	2393
other non-empty name	1421

Name Counts (FV3)

3078 transcript(s) had non-generic names.

"conserved hypothetical protein"	8913
"hypothetical protein"	628
"predicted protein"	1580
hypothetical protein similar to...	1921
other non-empty name	1157

Name Counts (FG3)

3290 transcript(s) had non-generic names.

"conserved hypothetical protein"	6368
"hypothetical protein"	607
"hypothetical protein "	41
"predicted protein"	3067
hypothetical protein similar to...	1980
other non-empty name	1269

Gene Numbering

Every annotated gene is given a Locus Number of the form FV3G_##### that should be considered the only guaranteed way to identify a gene uniquely. Each locus number is guaranteed to identify a unique gene even over different assemblies. Loci are simply identifiers and are not guaranteed to have any particular order or internal structure. We feel that it is a bad idea to encode attributes of an object, such as position, in its identifier. Position is an attribute of a gene that can be retrieved by the locus.

Overview of Query Genes

Query Genes (FO2)

17735 genes (8847 on '+' strand, 8888 on '?')
17735 transcripts (13331 spliced, 4404 unspliced)
47896 exons, 30161 introns

	len	%cov	%gc	%at
genic	26717541	44.58	50.73	49.27
intergenic	33219242	55.42	46.52	53.48
exonic	23693318	39.53	51.41	48.59
intronic	3015569	5.03	45.47	54.53
coding	21905719	36.55	51.88	48.12
5' UTR	773537	1.29	47.96	52.04
3' UTR	1171889	1.96	44.93	55.07
alt. spliced	8654	0.01	44.94	55.06
genomic	59936783	100.00	48.40	51.60
	min	median	mean	n50	max
total length (incl. UTR + introns)	90	1292	1519	1849	22869
coding length	90	1023	1236	1542	22596
exons per transcript	1	2	2.70	3	20
exons per spliced transcript	2	3	3.26	3	20
bp per exon	1	288	499	939	13941
bp per intron	4	58	101	129	2370
5' UTR bp	1	120	186	306	6664
3' UTR bp	1	194	279	391	5839

Query Genes (FV3)

14179 genes: 14199 transcripts (10878 spliced, 3321 unspliced; 7182/7017 +/-)
39508 exons, 25309 introns

	len	%cov	%gc	%at
genic	22749525	54.55	50.70	49.27
intergenic	18950820	45.45	46.31	53.73
exonic	20322204	48.73	51.33	48.67
intronic	2436142	5.84	45.44	54.26
coding	17889392	42.90	52.03	47.97
5' UTR	1071342	2.57	48.34	51.66
3' UTR	1519984	3.65	45.35	54.65
alt. spliced	8821	0.02	45.81	54.19
genomic	41700345	100.00	48.70	51.30
	min	median	mean	max
overall length (incl. UTR)	90	1239	1445	22650
coding length	90	1059	1260	22650
exons per transcript	1	2	2.78	21
exons per spliced transcript	2	3	3.33	21
bp per exon	1	303	519	22425
bp per intron	22	57	96	989
5' UTR bp	1	123	195	2994
3' UTR bp	1	191	277	3118

Query Genes (FG3)

13332 genes: 13332 transcripts (10278 spliced, 3054 unspliced; 6703/6629 +/-)
37575 exons, 24243 introns

	len	%cov	%gc	%at
genic	20999352	57.97	50.41	49.53
intergenic	15224289	42.03	45.47	54.62
exonic	18972091	52.37	51.14	48.83
intronic	2032901	5.61	43.53	56.05
coding	17874256	49.34	51.57	48.42
5' UTR	387612	1.07	46.54	53.44
3' UTR	799949	2.21	44.15	55.62
alt. spliced	5640	0.02	43.97	56.03
genomic	36223641	100.00	48.33	51.67
	min	median	mean	max
overall length (incl. UTR)	90	1206	1430	33594
coding length	90	1103	1340	33594
exons per transcript	1	2	2.82	21
exons per spliced transcript	2	3	3.36	21
bp per exon	1	283	507	29066
bp per intron	1	56	83	975
5' UTR bp	1	111	158	4754
3' UTR bp	1	190	263	3601

Overview of Reference genes

Reference Genes (FO2)

6568 genes (3278 on '+' strand, 4038 on '?')
7316 transcripts (3928 spliced, 3388 unspliced)
14174 exons, 6858 introns

	len	%cov	%gc	%at
genic	7096135	11.84	51.14	48.86
intergenic	52840648	88.16	48.03	51.97
exonic	6664716	11.12	51.51	48.49
intronic	389597	0.65	45.04	54.96
coding	5489634	9.16	52.54	47.46
5' UTR	574430	0.96	49.65	50.35
3' UTR	750471	1.25	45.28	54.72
alt. spliced	41822	0.07	48.48	51.52
genomic	59936783	100.00	48.40	51.60
	min	median	mean	n50	max
total length (incl. UTR + introns)	27	958	1097	1252	5036
coding length	27	763	826	918	4932
exons per transcript	1	2	1.94	2	10
exons per spliced transcript	2	2	2.75	5	10
bp per exon	4	422	530	805	4541
bp per intron	20	55	75	63	782
5' UTR bp	1	67	116	247	1897
3' UTR bp	1	86	153	326	2411

Reference Genes (FV3)

9522 genes: 10855 transcripts (6669 spliced, 4186 unspliced; 5455/5400 +/-)
24590 exons, 13735 introns

	len	%cov	%gc	%at
genic	11182255	26.82	50.57	49.43
intergenic	30518090	73.18	48.02	51.98
exonic	10465540	25.10	50.94	49.06
intronic	805534	1.93	45.41	54.55
coding	8913639	21.38	51.95	48.05
5' UTR	376175	0.90	47.25	52.75
3' UTR	1392140	3.34	45.23	54.77
alt. spliced	88819	0.21	47.96	52.04
genomic	41700345	100.00	48.70	51.30
	min	median	mean	max
overall length (incl. UTR)	79	957	1137	7912
coding length	79	849	944	7824
exons per transcript	1	2	2.27	21
exons per spliced transcript	2	3	3.06	21
bp per exon	3	378	501	4737
bp per intron	20	54	72	791
5' UTR bp	1	132	185	2994
3' UTR bp	1	182	257	3021

Reference Genes (FG3)

4714 genes: 5163 transcripts (2714 spliced, 2449 unspliced; 2623/2540 +/-)
10006 exons, 4843 introns

	len	%cov	%gc	%at
genic	4051152	11.18	50.66	49.34
intergenic	32172489	88.82	48.04	51.96
exonic	3758384	10.38	51.15	48.85
intronic	321070	0.89	44.58	55.39
coding	0	0.00	-	-
5' UTR	0	0.00	-	-
3' UTR	0	0.00	-	-
alt. spliced	28302	0.08	47.37	52.63
genomic	36223641	100.00	48.33	51.67
	min	median	mean	max
overall length (incl. UTR)	29	760	802	5079
coding length	-	-	-	-
exons per transcript	1	2	1.94	12
exons per spliced transcript	2	2	2.78	12
bp per exon	4	324	414	4244
bp per intron	24	56	76	767
5' UTR bp	-	-	-	-
3' UTR bp	-	-	-	-

Splice Analysis

Splice Analysis (FO2)

15205 splice agreements, 740 disagreements.
8566 ignored: 194 due to EST misalignment, 8372 due to partial initial/terminal exon coverage.
perfect exon:exon/intron:intron matches: 5159/5521
10 query transcripts contained noncanonical splices.

transcripts with no splice problems	5145	94.2%
... with complete reference coverage	791	14.5%
explainable by alternate splicing	212	3.9%
... with spliced reference	151	2.8%
clashes	102	1.9%
... with spliced reference	67	1.2%

Transcripts that have a splice site disagreement with an overlapping reference gene are placed into two categories, depending on the severity of the clash. If all splice disagreements could be explained by well-known types of alternate splicing, we call the transcript a "possible alternate splice." If the two transcripts cannot be reconciled in this way, we label the query a "clash." In partitioning splice disagreements into two categories, we are not asserting that 3.9% of this genome shows alternate splicing. We do this as a form of triage: genes in the "clash" category are manually inspected before release.

		in ref.	in query
cassette exons		9	0
retained introns		123	28
early 3' splices		6	50
late 5' splices		8	26

cassette exonan exon that falls completely with an intron of a variant transcript. Such exons may represent alternative splice forms but are more likely instances of exonic over- and under-prediction. retained intronan intron that falls within the exon of a variant transcript. These introns may indicate alternative splicing but usually are over- and under-predicted introns. early 3' splicestwo introns agree on their 5' splice site but differ on the 3' side, relative to the affected intron. In other words, differing 3' splice sites lie on the leading edge of an exon. late 5' splicestwo introns agree on their 3' splice site but differ on the 5' side, again relative to the affected intron. Most terminology is from Matlin AJ, et. al. Understanding alternative splicing: towards a cellular code. Nat Rev Mol Cell Biol. 2005 May;6(5):386-98.

Splice Analysis (FV2)

32001 splice agreements, 1492 disagreements.
6873 ignored: 112 due to EST misalignment, 6761 due to partial initial/terminal exon coverage.
perfect exon:exon/intron:intron matches: 13696/10397
0 query transcripts contained noncanonical splices.

transcripts with no splice problems	7089	92.0%
... with complete reference coverage	3783	49.1%
explainable by alternate splicing	553	7.2%
... with spliced reference	443	5.7%
clashes	66	0.9%
... with spliced reference	55	0.7%

Transcripts that have a splice site disagreement with an overlapping reference gene are placed into two categories, depending on the severity of the clash. If all splice disagreements could be explained by well-known types of alternate splicing, we call the transcript a "possible alternate splice." If the two transcripts cannot be reconciled in this way, we label the query a "clash." In partitioning splice disagreements into two categories, we are not asserting that 7.2% of this genome shows alternate splicing. We do this as a form of triage: genes in the "clash" category are manually inspected before release.

		in ref.	in query
cassette exons		27	2
retained introns		56	235
early 3' splices		16	189
late 5' splices		28	96

cassette exonan exon that falls completely with an intron of a variant transcript. Such exons may represent alternative splice forms but are more likely instances of exonic over- and under-prediction. retained intronan intron that falls within the exon of a variant transcript. These introns may indicate alternative splicing but usually are over- and under-predicted introns. early 3' splicestwo introns agree on their 5' splice site but differ on the 3' side, relative to the affected intron. In other words, differing 3' splice sites lie on the leading edge of an exon. late 5' splicestwo introns agree on their 3' splice site but differ on the 5' side, again relative to the affected intron. Most terminology is from Matlin AJ, et. al. Understanding alternative splicing: towards a cellular code. Nat Rev Mol Cell Biol. 2005 May;6(5):386-98.

Splice Analysis (FG3)

11676 splice agreements, 590 disagreements.
4631 ignored: 90 due to EST misalignment, 4541 due to partial initial/terminal exon coverage.
perfect exon:exon/intron:intron matches: 4246/4024
2 query transcripts contained noncanonical splices.

transcripts with no splice problems	3550	93.5%
... with complete reference coverage	886	23.3%
explainable by alternate splicing	156	4.1%
... with spliced reference	105	2.8%
clashes	91	2.4%
... with spliced reference	43	1.1%

Transcripts that have a splice site disagreement with an overlapping reference gene are placed into two categories, depending on the severity of the clash. If all splice disagreements could be explained by well-known types of alternate splicing, we call the transcript a "possible alternate splice." If the two transcripts cannot be reconciled in this way, we label the query a "clash." In partitioning splice disagreements into two categories, we are not asserting that 4.1% of this genome shows alternate splicing. We do this as a form of triage: genes in the "clash" category are manually inspected before release.

		in ref.	in query
cassette exons		2	0
retained introns		97	16
early 3' splices		13	25
late 5' splices		5	16

cassette exonan exon that falls completely with an intron of a variant transcript. Such exons may represent alternative splice forms but are more likely instances of exonic over- and under-prediction. retained intronan intron that falls within the exon of a variant transcript. These introns may indicate alternative splicing but usually are over- and under-predicted introns. early 3' splicestwo introns agree on their 5' splice site but differ on the 3' side, relative to the affected intron. In other words, differing 3' splice sites lie on the leading edge of an exon. late 5' splicestwo introns agree on their 3' splice site but differ on the 5' side, again relative to the affected intron. Most terminology is from Matlin AJ, et. al. Understanding alternative splicing: towards a cellular code. Nat Rev Mol Cell Biol. 2005 May;6(5):386-98.

Possible Problems

Problems-Fusarium oxysporum (FO2)

		FO2_GENETRANSFER_NT_FG1_MIPS_GENES									24
			FO2_GENETRANSFER_FG1_MIPS_GENES								372
				FO2_GENETRANSFER_NT_FV3_1							27
					{multiple sources}						8884
						FO2_GENETRANSFER_FV3_6					273
							FO2_FGENESH_1				4812
								FO2_GENEID_1			3284
									FO2_MANUAL_1		59
short proteins < 50aa	134	1	0	0	28	1	18	86	0	<-- not tallied in problems
shorter proteins < 30aa	0	-	-	-	-	-	-	-	-
very short proteins < 10aa	0	-	-	-	-	-	-	-	-
initial exon ≤ 6bp	181	0	1	1	49	1	92	37	0
internal exon ≤ 6bp	61	0	0	0	18	0	43	0	0
terminal exon ≤ 6bp	120	0	0	0	13	0	54	53	0
≥ 15 exons	7	0	0	0	2	0	2	2	1
intron ≥ 1000bp	4	0	0	0	0	1	3	0	0
intron ≤ 20bp	5	0	1	0	4	0	0	0	0
first codon not Met	170	1	63	0	81	23	0	2	0	<-- not tallied in problems
first codon not xTG	145	1	59	0	63	20	0	2	0
first codon not known START	155	1	59	0	71	22	0	2	0
last codon not STOP	220	5	76	3	97	25	9	4	1
contains in-frame STOP	0	-	-	-	-	-	-	-	-
coding length not modulo 3	173	9	2	8	139	10	5	0	0
non-canonical splicing	110	0	1	4	92	7	0	0	6
has ≥1 good BLAST hit	12437	21	269	19	7127	191	3208	1553	49	<-- not tallied in problems
≤1/3 as long as BLAST hit	296	0	3	1	37	3	115	136	1
≥3? longer than BLAST hit	38	0	3	0	19	0	11	5	0
contains ≥1 N in exon	40	3	5	2	21	7	0	0	2
contains low-quality sequence	1742	3	71	3	597	116	615	327	10	<-- not tallied in problems
touches gap(s)	190	3	10	2	52	16	103	0	4
spans contigs	179	3	9	2	44	15	103	0	3
within 1kb of contig edge	1183	5	34	3	356	33	408	336	8	<-- not tallied in problems
any overlap (UTR or CDS)	309	0	7	0	493	15	59	48	8	<-- in 309 clusters
CDS overlap only	0	-	-	-	-	-	-	-	-
CDS overlap > 50bp	0	-	-	-	-	-	-	-	-
CDS overlap > 100bp	0	-	-	-	-	-	-	-	-
CDS overlap > 200bp	0	-	-	-	-	-	-	-	-
has predicted UTR	5056	4	40	12	4518	119	138	171	54	<-- not tallied in problems
UTR ≥ 50% length	503	0	1	0	454	7	5	30	6	<-- not tallied in problems
UTR is spliced	413	0	3	1	354	8	10	28	9	<-- not tallied in problems
one or more problems	1886	12	113	15	913	68	467	279	19

Problems-Fusarium verticillioides (FV3)

		{multiple sources}						1917
			FV3_FINDORFS_6					3127
				FV3_FGENESH_1				4468
					FV3_MANUAL_1			1029
						FV3_GENEID_4		3658
short proteins < 50aa	166	0	0	14	56	96	<-- not tallied in problems
shorter proteins < 30aa	0	-	-	-	-	-
very short proteins < 10aa	0	-	-	-	-	-
initial exon ≤ 6bp	130	7	0	81	0	42
internal exon ≤ 6bp	44	0	7	35	1	1
terminal exon ≤ 6bp	74	0	0	34	0	40
≥ 15 exons	11	0	2	4	2	3
intron ≥ 1000bp	0	-	-	-	-	-
intron ≤ 20bp	0	-	-	-	-	-
first codon not Met	7	0	0	0	6	1	<-- not tallied in problems
first codon not xTG	7	0	0	0	6	1
first codon not known START	7	0	0	0	6	1
last codon not STOP	12	0	0	1	10	1
contains in-frame STOP	4	0	0	0	4	0
coding length not modulo 3	13	0	0	0	13	0
non-canonical splicing	0	-	-	-	-	-
has ≥1 good BLAST hit	11262	1629	2889	3609	654	2481	<-- not tallied in problems
≤1/3 as long as BLAST hit	263	4	7	75	63	114
≥3? longer than BLAST hit	68	6	4	37	6	15
contains ≥1 N in exon	0	-	-	-	-	-
contains low-scoring sequence	367	29	78	138	36	86
touches gap(s)	26	0	1	20	4	1
spans contigs	26	0	1	20	4	1
within 1kb of contig edge	167	20	27	53	23	44	<-- not tallied in problems
any overlap (UTR or CDS)	382	78	304	161	103	127	<-- in 382 clusters
CDS overlap only	0	-	-	-	-	-
CDS overlap > 50bp	0	-	-	-	-	-
CDS overlap > 100bp	0	-	-	-	-	-
CDS overlap > 200bp	0	-	-	-	-	-
has predicted UTR	6567	670	3123	858	982	934	<-- not tallied in problems
UTR ≥ 50% length	924	65	317	52	382	108	<-- not tallied in problems
UTR is spliced	752	55	233	63	246	155	<-- not tallied in problems
one or more problems	1686	122	389	549	217	409

Problems-Fusarium graminearum (FG3)

		FG3_GENETRANSFER_FG1_MIPS_GENES									4327
			FG3_GENETRANSFER_FV3_GENES								246
				FG3_GENETRANSFER_AA_FV3_1							117
					{multiple sources}						6269
						FG3_FL_EST_GENES					23
							FG3_FGENESH_1				811
								FG3_GENEID_1			1460
									FG3_MANUAL_1		79
short proteins < 50aa	53	4	2	2	5	0	3	32	5	<-- not tallied in problems
shorter proteins < 30aa	0	-	-	-	-	-	-	-	-
very short proteins < 10aa	0	-	-	-	-	-	-	-	-
initial exon ≤ 6bp	126	58	5	0	26	0	20	16	1
internal exon ≤ 6bp	68	48	2	0	8	0	9	0	1
terminal exon ≤ 6bp	69	32	1	0	2	0	7	27	0
≥ 15 exons	13	8	0	0	1	0	1	2	1
intron ≥ 1000bp	0	-	-	-	-	-	-	-	-
intron ≤ 20bp	145	144	0	1	0	0	0	0	0
first codon not Met	25	8	8	1	2	0	0	1	5	<-- not tallied in problems
first codon not xTG	24	7	8	1	2	0	0	1	5
first codon not known START	24	7	8	1	2	0	0	1	5
last codon not STOP	34	11	9	8	1	0	0	3	2
contains in-frame STOP	2	0	0	0	0	0	0	0	2
coding length not modulo 3	18	4	9	0	2	0	0	0	3
non-canonical splicing	23	15	7	0	0	0	0	0	1
has ≥1 good BLAST hit	9446	2487	225	103	5284	15	708	576	48	<-- not tallied in problems
≤1/3 as long as BLAST hit	16	5	2	0	1	0	1	5	2
≥3? longer than BLAST hit	41	19	0	0	9	0	8	5	0
contains ≥1 N in exon	10	2	7	0	0	0	0	0	1
contains low-quality sequence	311	65	19	11	128	1	55	28	4	<-- not tallied in problems
touches gap(s)	70	2	8	0	7	0	50	1	2
spans contigs	67	1	8	0	5	0	50	1	2
within 1kb of contig edge	479	119	15	6	163	0	81	83	12	<-- not tallied in problems
any overlap (UTR or CDS)	256	91	25	3	340	1	19	26	13	<-- in 256 clusters
CDS overlap only	0	-	-	-	-	-	-	-	-
CDS overlap > 50bp	0	-	-	-	-	-	-	-	-
CDS overlap > 100bp	0	-	-	-	-	-	-	-	-
CDS overlap > 200bp	0	-	-	-	-	-	-	-	-
has predicted UTR	3359	333	167	6	2549	23	83	123	75	<-- not tallied in problems
UTR ≥ 50% length	437	26	32	1	319	1	4	32	22	<-- not tallied in problems
UTR is spliced	222	17	20	1	144	0	2	23	15	<-- not tallied in problems
one or more problems	1078	403	52	12	395	1	106	85	24