ncyc's People
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viridianaavila jianshu93 liaohu1231 chlige jameyzhu xty888 luanxiao carden24 25280841 liaochenlanruo wjwang20220505 sibo-zhang taichu001 kaitlin-rempfert microbiomecat anyihu labhanmz chuang2016 chengzhuangchen hj1994412 anananxiaochi utguangncyc's Issues
Incorrect parameters
what's the difference between the "NCyc_100_2019Jul" and "NCyc_100.faa"?
As mentioned in the title, I want to annotate the N genes in some MAGs, if I can use the ORF predicted from prodigal? And I want to know the difference between the two databases. Before use, are they must be unzipped?
comammox amoA?
Hello
I am interested in how many comammox amoA genes are in my samples, but I don`t know whether this database contain these genes. If contains, at this time can I separate them from other amoA_A genes ?
thanks
hu
more power
Hello! Can I assign more threads to process? Its using only one processor.
problem using code
Can I combine output from multiple runs?
Can output from different runs be combined or is the abundance relative to all other present samples?
Can I specify a single input for -d so I can run in parallel
I have 3 large assemblies and would rather be allowed to run the in parallel. Is this allowed or must they be run together?
-d : specify the directory where your fasta/fastq (or gzipped) files are located.
Getting the annotated gene/protein sequence
Hi,
Thanks for the great database. I used NCYC on my MAG's contig and got the results. one thing I want to make sure is to get the particular gene sequence used for annotation. For say,
G01_000000067461 0fd91084f91d2de2731de1f1356dd7d6 nirB
In the above result, it says G01_000000067461 corresponds to nirB gene, but in reality, G01_000000067461 is a part of a contig/scaffold representing multiple genes. So how do I can get the particular gene sequence from G01_000000067461 used by NCYC for nirB.
Similarly, the diamond output file does not have any headers, and it's a bit confusing to what some of the number and characters correspond to.
Diamond output
G01_000000067461 0fd91084f91d2de2731de1f1356dd7d6 80.6 813 155 2 69690 67258 1 812 0.0e+00 1329.3
Bug with system call 'blastall'
Hi All,
I'm afraid blastall -d
flag only receives custom-built or NCBI standard database, e.g. nr, refseq.
Raw fasta files must be built into a custom blast database in prior for blastp
.
But in the line 77 of NCycProfiler.PL
:
system("blastall -p blastp -d data/NCyc_100.faa -i $file -o $out -m 8 -e 1e-4 -b 1 -v 1")
attempts to directly assign the faa file to the -d
flag. Has anyone made this successful?
BTW, the use of blastall
is also discontinued from legacy blast suite. For users using newer versions of blast, modify the script to use blastp/blastn
instead.
In addition, the error message of the above error is confusing and not documented. Since no return value checked after the system
call, the fail of blastall
is not caught. The script keeps execution and ended up in an error message from cp
instead: no such file or directory of some_sample.blast (due to no output from blastall).
-m diamond
has no this issue. Not checked with usearch mode.
Question about NCyc_95.faa.gz vs NCyc_100.faa.gz
Hello and thank you for this very useful tool for finding N-cycling related genes!
I have a question regarding the output of using the NCyc_95 vs NCyc_100(_2019Jul) databases: how is it that the 95 database can produce more hits for a gene than the 100 database? To provide some background, I am running the program on a set of MAGs and wanted to see how the output would differ using the two different databases. I have multiple MAGs where there are a greater number of hits for genes resulting from using the 95 database compared to the 100 database. Why does this happen?
I am curious about this because it would seem that any of the representative sequences belonging to the 95 database should also be present in the 100 database? Am I misunderstanding how these two databases are created?
I am running the tool using Diamond and am happy to share any code or resulting output of the program as you may need! Thank you for your time and advice!
Accession IDs of reference database
Hi,
Is it possible to provide the accession IDs of the sequences used in NCyc_100.faa.gz?
Thanks,
Ruben
Script usage example
Hi,
Thanks for this great work! I'm interested in using it.
Could you please provide a short example on how to use the NCycProfiler.PL
script for profiling metagenomic sequence data (e.g., fasta of gene sequences).
Thanks,
Ruben
How do you generate the count tables?
I assume you parse the diamond output to somehow generate the count tables? I am wondering how you do this as I would like to know on what contigs the genes were found so I can generate a pseudo abundance of that gene based on mapping depth to that contig.
I wish to do this as a count is only telling me how contigs with that gene on exist but hides the potential biological significant of how abundant that contig may be (i.e. how abundant that bacteria it came from it)
Format issue with -si file
Hi,
I have a similar problem to issue #11. I am running NCyc on two samples as a test. These files are called sample31merged.fastq.gz (127189806 reads)and sample32merged.fastq.gz (191219006 reads).
I run the following command:
perl NCycProfiler.PL -d ~/Dorset_mesocosms/ProjectLIMS19618/CLEAN_READS2/MERGED_READS/ncyctest/ -m diamond -f fastq.gz -s nucl -si ~/Dorset_mesocosms/ProjectLIMS19618/CLEAN_READS2/MERGED_READS/ncyctest/samples.tsv -o ~/nitro_genes.tsv
After running I get the following:
Deallocating buffers... [0.043s]
Deallocating queries... [0.004s]
Loading query sequences... [0s]
Closing the input file... [0.018s]
Closing the output file... [0.132s]
Closing the database file... [0.01s]
Deallocating taxonomy... [0s]
Total time = 5829.35s
Reported 3850528 pairwise alignments, 3850528 HSPs.
3850528 queries aligned.
The host system is detected to have 1621 GB of RAM. It is recommended to increase the block size for better performance using these parameters : -b12 -c1
was not found in /home/bgregs/Dorset_mesocosms/ProjectLIMS19618/CLEAN_READS2/MERGED_READS/ncyctest/samples.tsv, please check!
Diamond appears to successfully run generating sample31merged.diamond and sample32merged.diamond files but then I get the above error and there is no further output. This seems to be an issue with reading the sample information file.
I've tried a few different formats with the sample info file. I have tried it with (samples.tsv) or without (samples2.tsv) the fastq.gz extension as recommended in issue #14 and without the full file path (sampleinfo.tsv) but I get the same error regardless. I have attached these files as .txt files as I can't upload tsv to GitHub:
sampleinfo.txt
samples2.txt
samples.txt
The only changes I have made to the script is path to diamond:
##Please specify where your prefered database searching tool locates
##NCBI blast ftp://ftp.ncbi.nlm.nih.gov/blast/executables/legacy.NOTSUPPORTED/2.2.26/blast-2.2.26-x64-linux.tar.gz
my $blast = "/bin/blast/bin/blastall";/bin/blast/bin/formatdb";
my $formatdb = "
##diamond https://github.com/bbuchfink/diamond/releases
my $diamond = "/usr/local/bin/diamond2";
##usearch https://www.drive5.com/usearch/download.html
my $usearch = "~/bin/usearch8.1.1861_i86linux32";
Any advice to sort out this issue would be appreciated.
error when making blast db
Hi!
I am trying to make a blast db with the NCyc100.faa.gz file (I also tried with the 95 and got the same error) so that I can blast it outside of the perl executable you provide. I basically unzip the file and then run makeblastdb, However I can't make the database because i get the following error:
`BLAST Database creation error: Error: Duplicate seq_ids are found:
LCL|39946.BGIOSGA039077-PA
`
Has this issue happened before?
Thank you!
Why do annotating the same functionality with different databases produce different results?
Hello,
I made functional annotation associated with nitrogen metabolism for metagenomic data using KofamScan(https://www.genome.jp/tools/kofamkoala/) ,NCyc and eggnog-mapper. But these results are significantly different. For example, the ORFs that are annotatated as gene nirK using three methods are different. Why is it so?
Thank you!
-si file: There is always a missing sample name after alignment ending
Dear @qichao1984
Thanks for the NCyc. This is me asking for guidance.
I'm using your script as follows:
$ perl NCycProfiler.pl -d ~/sickle/. -m diamond -f fastq -s nucl -si ~/sickle/samples.tsv -o ~/nitro_genes.tsv
After running, i get the following:
[...] Loading reference sequences... [1.2e-05s]
Deallocating buffers... [0.014192s]
Deallocating queries... [0.028869s]
Loading query sequences... [1e-05s]
Closing the input file... [7e-06s]
Closing the output file... [4.3e-05s]
Closing the database file... [5e-06s]
Deallocating taxonomy... [3e-06s]
Total time = 150.442s
Reported 153742 pairwise alignments, 153742 HSPs.
153742 queries aligned.
RP_IV_2 was not found in ~/sickle/samples.tsv, please check!
I tried running just two samples (two file names with their seqs counts in samples.tsv
), though I got the same ¡error!
Information about samples.tsv
--> I have 10 fastq files in ~/sickle/. Those fastq files are listed in samples.tsv
without the file extension, and their seq counts each. Values are \t separated.
So, Would you mind give a hand?
Thanks.
MaryoHg.
missing output file
I am running the script as follows:
perl /programmes/NCyc/NCycProfiler.pl c -d /prepost/final_assemblies/matriline_assembly/contig_500bp_filt/cd-hit_dereplication_output/metaProdigal_output -m diamond -f faa -s prot -si seqnum.txt -o /prepost/final_assemblies/matriline_assembly/contig_500bp_filt/cd-hit_dereplication_output/metaProdigal_output/ncyc_output/ncyc_profiles.txt
yet I am getting no output to ncyc_profiles.txt
. The run is executing correctly it seems and I do get the raw diamond output.
Any idea what I am doing wrong?
NcycDB license?
Hi,
I am interested in using NcycDB in my research but I see the Github repo does not contain a license for use of this code. I worry that means I cannot use the code in my research, because Github's support page says that no license means the code cannot be freely distributed.
Can you attach a license to this repo that allows for use of the code?
thanks for your time!
No diamond/usearch/blast files were detected!
Hi, I attempted to run NCycProfiler.PL as follows:
$ perl NCycProfiler.PL -d ~/data/ -m /usr/bin/usearch -f fq -s nucl -si sample_info.txt -o NCyc_output
No diamond/usearch/blast files were detected!
What usearch files is the script trying to detect? I thought the script is intended to run usearch against the provided *faa.gz files. Am I supposed to run usearch (or diamond or blast) on my selected *faa.gz file before running the script, ie is the script looking for the output of a usearch run?
Here are some details on my usearch version, sample info file and read files
$ /usr/bin/usearch10
usearch v10.0.240_i86linux64, 65.6Gb RAM, 32 cores
(C) Copyright 2013-17 Robert C. Edgar, all rights reserved. http://drive5.com/usearch
License: [redacted], non-profit use, max 1 process(es)
$ head -2 sample_info.txt
17 2608559
18 325473
$ ls data/ | head -2
17.fq
18.fq
EDIT: updated usearch location and version info
What is what in the output?
I am getting output files looking like this but have no idea what is what?
k119_1934631_1 bur:Bcep18194_A4430 30.5 174 112 4 2 169 176 346 1.5e-12 68.2
k119_1254898_1 693661.Arcve_0699 32.6 215 121 4 33 223 14 228 1.1e-18 90.5
k119_1150324_1 5911.EAS00354 39.8 472 240 8 47 510 34 469 7.3e-89 282.7
k119_575171_1 1001530.PMSV_1997 46.8 111 51 1 1 103 1 111 2.4e-28 104.0
k119_1882347_1 34dd61e2fef5739d26ce3de0c76f9d94 48.5 171 86 2 1 169 176 346 1.8e-49 168.7
k119_1150325_2 6d0871a2edb457b604224c7994886b06 43.2 111 60 3 3 112 460 568 3.0e-16 77.8
k119_1516336_1 60051142d5b017a88152f3baddc63d81 36.6 243 127 6 29 244 238 480 1.4e-34 134.0
k119_1359477_1 374308263 71.4 56 16 0 1 56 1 56 1.1e-11 55.5
k119_1673198_1 350688.Clos_0484 68.9 405 120 4 4 405 383 784 9.9e-181 538.5
k119_1464047_1 339323450 49.5 107 53 1 235 340 42 148 7.9e-26 103.2
k119_522884_1 431895.XP_001747511.1 32.8 299 178 6 76 352 642 939 1.5e-42 160.6
k119_575174_1 866774.HMPREF9248_0150 78.1 32 7 0 9 40 23 54 3.0e-10 54.7
k119_1830061_1 642492.Clole_3012 66.2 287 97 0 1 287 387 673 2.4e-133 399.4
k119_1411762_1 212717.CTC00060 52.6 578 249 6 1 562 188 756 8.1e-202 585.5
k119_418314_1 e29be0bd0da4c63f6c0a39033ee3db32 48.9 47 24 0 5 51 93 139 3.4e-09 52.8
k119_470601_1 5825.PCHAS_101490 28.0 157 80 9 5 131 12 165 3.9e-06 49.3
k119_1359482_2 526221.XP_003003854.1 30.6 121 79 3 2 121 21 137 1.3e-08 53.5
k119_1202611_2 116747712 24.1 261 158 8 374 614 591 831 1.1e-05 52.0
k119_1202611_3 411464.DESPIG_00203 24.9 706 349 21 3 590 2 644 3.5e-42 165.2
k119_418316_2 a2fc249a6799579a69695a177760985a 53.0 66 31 0 168 233 10 75 3.5e-15 70.9
k119_366024_2 15669255 40.3 139 81 1 3 139 243 381 1.1e-26 105.1
k119_1307192_3 d9870872203d1ba76e85b283d4febb26 36.5 115 62 3 76 190 138 241 3.5e-08 54.7
k119_627464_1 fee9685fb9783d0114adb8c48bcfbf19 50.0 58 29 0 144 201 336 393 1.9e-06 50.1
k119_1254909_1 B6GAA7 62.3 154 58 0 1 154 461 614 1.1e-69 223.4
k119_418318_1 1ba5484590e9b2f67f7d6547226cc24c 48.1 131 67 1 1 130 97 227 3.4e-39 133.7
The first column is contig name and then I don't know what the rest are. Would you be able to provide some insight please?
set up blast in the conda environment
Hi Professor Tu,
I set up blast using conda,
in the code,
##NCBI blast ftp://ftp.ncbi.nlm.nih.gov/blast/executables/legacy.NOTSUPPORTED/2.2.26/blast-2.2.26-x64-linux.tar.gz
(cannot get this gz file using this web)
my $blast = "~/bin/blast/bin/blastall";
(no blastall in bin folder, just have blastx, blastn, blastp in /home/lbhuang/miniconda3/pkgs/blast-2.12.0-pl5262h3289130_0/bin/)
my $formatdb = "~/bin/blast/bin/formatdb";
(no formatdb in bin folder)
so which one can I use?
Thank you very much
classification using database
It is possible to get the classification for some interesting functional genes:
I want to know both the abundance and the classification for AOA, AOB, nirK/S and nosZ; wonder how can I do this using this database and pipeline.
thank you
Is number of sequence just number of identified ORFs in the -si file?
Pretty much as title says. What is th enumber of sequences for the -si
file?
Secondly, do you have any suggestions of best practices for the -rs options?
Output file not detected
Hello,
I ran the perl script. The script runs till the following message is displayed:
Reported 91 pairwise alignments, 100 HSSPs
91 queries aligned.
#3
and terminates
I can't locate my output file. Can you mention where that might be.
Here is my script.
perl NCycProfiler.PL -d ~/prokka/cb2_bin9/ -m diamond -f fa -s nucl -si ~/prokka/cb2_bin9/ncyctry -o out_ncyc
Cannot find diamond output
Hi. :)
I am trying to run the following:
perl NCycProfiler.PL -d ~/Desktop/scripts_to_abacus/From_abacus/PC_AA_Seqeunces -m diamond -f fasta -s prot -si /home/christian/Bioinformatic_programs/NCyc-master/FileForNcyc.txt -o PC_Ncyc
However, I get the following error:
cp: cannot stat '/home/christian/Desktop/scripts_to_abacus/From_abacus/PC_AA_Seqeunces/AAsequencesF10786.diamond': No such file or directory
cp: cannot stat '/home/christian/Desktop/scripts_to_abacus/From_abacus/PC_AA_Seqeunces/AAsequencesF10787.diamond': No such file or directory
cp: cannot stat '/home/christian/Desktop/scripts_to_abacus/From_abacus/PC_AA_Seqeunces/AAsequencesF10788.diamond': No such file or directory
cp: cannot stat '/home/christian/Desktop/scripts_to_abacus/From_abacus/PC_AA_Seqeunces/AAsequencesF10789.diamond': No such file or directory
cp: cannot stat '/home/christian/Desktop/scripts_to_abacus/From_abacus/PC_AA_Seqeunces/AAsequencesF10790.diamond': No such file or directory
cp: cannot stat '/home/christian/Desktop/scripts_to_abacus/From_abacus/PC_AA_Seqeunces/AAsequencesF10791.diamond': No such file or directory
cp: cannot stat '/home/christian/Desktop/scripts_to_abacus/From_abacus/PC_AA_Seqeunces/AAsequencesF10792.diamond': No such file or directory
cp: cannot stat '/home/christian/Desktop/scripts_to_abacus/From_abacus/PC_AA_Seqeunces/AAsequencesF10793.diamond': No such file or directory
cp: cannot stat '/home/christian/Desktop/scripts_to_abacus/From_abacus/PC_AA_Seqeunces/AAsequencesF10794.diamond': No such file or directory
cp: cannot stat '/home/christian/Desktop/scripts_to_abacus/From_abacus/PC_AA_Seqeunces/AAsequencesF10795.diamond': No such file or directory
cp: cannot stat '/home/christian/Desktop/scripts_to_abacus/From_abacus/PC_AA_Seqeunces/AAsequencesF10796.diamond': No such file or directory
cp: cannot stat '/home/christian/Desktop/scripts_to_abacus/From_abacus/PC_AA_Seqeunces/AAsequencesF10797.diamond': No such file or directory
was not found in /home/christian/Bioinformatic_programs/NCyc-master/FileForNcyc.txt, please check!
My tab delimited file looks like this:
head FileForNcyc.txt
AAsequencesF10786 18190
AAsequencesF10787 10828
AAsequencesF10788 41144
AAsequencesF10789 44519
AAsequencesF10790 874
AAsequencesF10791 81255
AAsequencesF10792 61666
AAsequencesF10793 79880
AAsequencesF10794 63433
AAsequencesF10795 51039
Maybe, the problem is trivial, however I cant see how to fix it? :)
Also, I tried with usearch, got the same problem
Cheers
Addition of HMMER/HMMSEARCH tool?
I notice all the tools available here are alignment based. Would it not improve accuracy to use a HMMER/HMMSEARCH
tool give this is metagenomic?
Secondly, might I suggest differentiating the working directory from the input directory? Essential add an option to direct where the NCyc
database is stored?
Can Ncyc be used on eukaryotic genomes?
I have used this on my metagenomic data but also want a profile for the host. Is this tool usable on eukaryotic genomes?
Unsure how to get started...
Hi there,
Could you please walk me through how to use this tool? I'm working on Mac Catalina and just installed perl.
Should I copy/paste the perl script into my terminal? Do I need to pre-download Diamond?
Many thanks!
Does it suitable for plant
Hello, teacher, I happened to see this tool developed by you, which is similar to the direction I want to do, but what I do is nitrogen metabolism of plants. I would like to ask, is this tool and data set suitable for my plant research?
Thanks for your reply.
Where did my single hit of amoA_B go when my samples concatenated?
Dear @qichao1984,
Thanks heaps for this DB.
We observed the following when analyzing our samples.
We have 10 assemblies (*.fa files) with different numbers of contigs.
I ran the NcyPerl.pl on my 10 assemblies (using the below command) and the obtained results contained a single hit with amoA_B in one sample (Figure 1).
$ NCycProfiler.pl -d ./ -m diamond -f fa -s nucl -si si.txt -rs 33175 -o out_file 2>&1 | tee log.run1
Figure 1: output file using all samples (n = 10) at the same depth (random sampling to 33,175)
However, after running NCycProfiler.pl on all my assemblies concatenated (sum of all contigs, around half a million), that hit is gone (Fig. 2). I cat my *.fa and run the script as shown below.
$ cat $(ls *.fa) >> cat_assembly.fa
$ NCycProfiler.pl -d ./ -m diamond -f fa -s nucl -si si.txt -o out_file 2>&1 | tee log.cat
Fig. 2. Output file using all samples (n = 10) concatenated (sum of all contigs)
We appreciate your time and response to this.
Greatings,
MaryoHg.
DNA sequence
Cause I need to use the DNA seq of the N-related proteins, Can you publish the DNA sequence of each protein sequence?
Output file
diamond error "Please specify the location of diamond! at NCycProfiler.PL line 42."
I write your codes as following:
perl NCycProfiler.PL -d /home/lbertrans/ -m diamond -f fastq -s nucl -si Sequences_samples -o N_genes
The error is:
Please specify the location of diamond! at NCycProfiler.PL line 42.
I changed the path line 11:
my $diamond = "~/home/spack/opt/spack/linux-centos7-broadwell/gcc-4.9.4/diamond-2.0.11-obtc4vtc4dbd7irps2tz2jxstc4icaq5/bin/diamond"
It didn't work. I had the same error as before.
We are working in cluster server. Could it be the access to diamond software?
Thanks.
Lluís
Identifiers missing from id2gene.map.2019Jul
Hello,
Thanks for providing this tool! I noticed that there are a number of proteins in NCyc_100_2019Jul.faa missing from id2gene.map.2019Jul (273,501 proteins vs. 219,091 entries in mapping file). Is this intentional?
Jeff
Mapping_file issues
Hello!
According to the number of the second column in the output of the "NCycProfiler.PL" script, not all the annotation informations were found in the mapping file. How did this happen? (Software: usearch, database: ncyc_100_2019jul.faa, mapping file: id2gene.map.2019jul).
Thanks!
diamond question
I read the NCycProfiler.PL, it seems that can align gene sequences to NCycDB database. But the sseqid (Subject Seqid) result of diamond blastx is not the protein ID in NCycDB (e.g. A0A254MU54). The sseqid (Subject Seqid) result of diamond blastx is a long string of values (e.g. 312309.VF). Is there any step I'm missing or how do I match the sseqid (Subject Seqid) of diamond blastx to the protein ID of NCycDB. Thanks!
Two files found as same file and not in si
Hi!
Thanks for creating this useful tool.
I have an error that I cant solve, even using the new script uploaded 20 days ago.
My command is
perl NCycProfiler2.PL -d . -m diamond -f .faa -s prot -si sampleinfi.txt -o output14
and apparenty eveything tus fine but this is the last lines of the file:
Total time = 917.505s
Reported 698857 pairwise alignments, 698891 HSPs.
698857 queries aligned.
cp: './proteins_aa14diamond' and './proteins_aa14diamond' are the same file
was not found in sampleinfi.txt, please check!
this is my -si
sampleinfi.txt
and the files I have in the folder with the script file are:
data NCycProfiler2.PL proteins_aa14.faa
diamond NCycProfiler.PL README.md
diamond_manual.pdf LICENSE proteins_aa14diamond sampleinfi.txt
Can you please, please help me?
Thanks in advance,
Ed
Three potential inaccurate issues to be confirmed
Hi! While the using of NCycDB, I found some potential inaccurate issues in it. So I want to confirm them here from the author for further improvement of this useful database.
-
There are 441 duplicated IDs (and sequence) in the file NCyc_100.faa. For your information, ten duplicated IDs are listed below:
1000565.METUNv1_00600
100226.SCO5525
1002339.HMPREF9373_0600
1002339.HMPREF9373_0712
1003195.SCAT_4357
1004836.SXCC_03526
1006006.Mcup_0918
1006551.KOX_23300
1006551.KOX_27050
1026882.MAMP_00572 -
There are 81 duplicated IDs in the file id2gene.map, and what's more, the classification of anyone of these IDs are not exactly the same. Ten of the mentioned IDs are listed below for your information.
A0A060I505 napA
A0A060I505 nasA
A0A0D6FVV7 narG
A0A0D6FVV7 narZ
A0A127KYU8 nasB
A0A127KYU8 nirB
A0A142WVR2 narB
A0A142WVR2 nasA
A0A142Y6U0 narB
A0A142Y6U0 nasA
A0A142YH98 narB
A0A142YH98 nasA
A0A145CM87 narB
A0A145CM87 nasA
A0A1D8AZ75 narB
A0A1D8AZ75 nasA -
The ID list should be identical between the two files NCyc_100.faa and id2gene.map. But 54410 IDs in file NCyc_100.faa can not found in file id2gene.map, and 99288 IDs in file id2gene.map can not found in file NCyc_100.faa. The issue will cause inaccuracy when calculating the relative abundance of Nitrogen cycling genes (sub)families.
N-genes counts from contigs normalized by total reads is not good indicator of gene abundance
Should short reads from metagenomic data be mapped to those genes (or protein) (>90% identity for blastx or diamond blastx for example) that were found in the database to calculate relative abundance of each gene? but not only how many hits of that gene you have against the database because some shot reads may not be assembled successfully but they are actually part of those genes. I do not see any discussion about this in the SBB paper.
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