The original text is as follows：
3.3.2 Detecting TE insertions using paired-end FASTQ file as input
$ perl user_installed_path/ERVcaller_v.1.4.pl -i TE_seq -f .fq.gz -H hg38.fa -T TE_consensus.fa -I folder_of_input_data -O folder_for_output_files -t 12 -S 20 -BWA_MEM
如果使用了-BWA_MEM 参数会导致程序把输入文件当成bam文件，需要去掉-BWA_MEM程序才能正常运行

Hi!

Although I am getting a vcf with HERV-K insertions after running ERVcaller with some bam files (sorted by coordinate and with duplicates marked), I still have the sampleID_temp directory. Shouldn't this directory be erased when the program finishes?

In addition, I have been looking the logs and I get some errors in different samples:

One of the errors is this:

Argument "-" isn't numeric in subtraction (-) at ERVcaller_v.1.3.pl line 1305, <OUT3> line 2.

In another sample, I get this error:

Error in `[<-.data.frame`(`*tmp*`, , 2, value = NA_real_) :
  replacement has 1 row, data has 0
Calls: [<- -> [<-.data.frame
Execution halted

Do you know how can I solve this?

By the way, I am still getting the output vcf in these samples, so I was wondering if maybe I could ignore it.

Thanks!

Dear ERVcaller team,

Hi, I'm Oh.

Thanks for your program.

Then, I have a question.

Can I use GATK pre-processed bam files for input bam of ERVcaller?

I have only pre-processed bam files. In this text, pre-processing means sorting, marking duplicate reads, and recalibrating (https://gatk.broadinstitute.org/hc/en-us/articles/360035535912-Data-pre-processing-for-variant-discovery).

Many Thanks.

Oh.

Hello, Dr. Chen, I have the following problems when using the test data. May I ask how I can solve them? (I have the same problems when I use my own data.)

Hello, Dr. Chen~! I met a problem when I tried to run test data. The .err log showed me the detailed infor below:

and here was my command line:

how could I solve these problems?
thank you for your help!

Hi chenxu：
Does Hydra-Version-0.5.3 is required by ERVcaller？It seem that 2.2 and 3.1 is conflicting in User Manual of ERVcaller v1.4.

Hello, Dr. Xun Chen,
My target plant species has 26 chromosomes, the genome size of which is about 2.2 Gb. We name the chromosomes like chr01, chr02, chr03...chr24, chr25, chr26. I have run the whole pipeline for 139 individuals, however, I found that the final vcf file doesn't have calls for chromosomes form chr01 to chr09. Are there any naming rules for chromosome ID, or is this software useful for other species like plants?
All the best,
Rain

这是我用的命令：
perl /vol6/home/quluj/zt/software/ERVcaller_v1.4/ERVcaller_v1.4.pl \
-i MD4 \
-f .fastq.gz \
-H /vol6/home/quluj/zt/pkduck_ref/PK_ref.fa \
-T /vol6/home/quluj/zt/DUCK/teseq/LTR.fasta \
-t 12 -S 20 -G
但是每次都到第三步就报错，只能得到一个空的vcf文件。
Step 3: Validation...

Converting SAM to BAM file, and then Sort and index the BAM file......

[bam_sort_core] merging from 11 files and 11 in-memory blocks...
[bwa_index] Pack FASTA... [bns_fasta2bntseq] Failed to allocate 0 bytes at bntseq.c line 303: Success
[E::bwa_idx_load_from_disk] fail to locate the index files
[E::bwa_idx_load_from_disk] fail to locate the index files

Hello Dr.Chen,

I'm wondering if the ERVcaller could be used to input long-read sequencing? ( ..and any suggestion about how to adjust command parameters and any validation result of identification accuracy while applying TGS data?

Thank you~

Hello,

I gave the program a fastq as an input but during the preocess I got this problem:

gzip: ERR2304551_picard_sorted_soft.fastq.gz: unexpected end of file

How could I fix it?

Thank you!

Dear ERVcaller team.

Hi I'm Oh.

I have executed your program.
And I get output file, and output directory.

When I use your test bam, I have only one output file (TE_seq.vcf), and one temp directory.

However, when I use my sample bam, I get one more file.

--> 2016000051.vcf (Expected), 2016000051_h1_sorted.bam (No expected)

What is "_h1_sorted.bam" file?

thank you for developing great tool.
I had some final vcf files.
I didn't find additional filters and others filters.
So, status of detected TE : 0 to 5, type 0 is it ok?
and Are chr and position in vcf breaking point?
I don't understand where is breaking point, what are START & END means.
for example,
#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT TE_seq
chr1 5617379 . T <INS_MEI:HERV>
. . TSD=NULL,NULL;INFOR=HERVK, 1,7831,7831,+,4;CR=64;SR=3;GTF=YES;GR=1.000 GT:GQ:GL:DPN:DPI
1/1:40:0,0,1:0:67

where is HERVK's insertion position?

你好，陈博，readme里面写到本软件会包含SE_MEI的脚本，但是我并没有看到。
于是我到SE_MEI原作者的GitHub里面下载安装了SE_MEI（改了htslib的版本为1.1），可以跑通ERVcaller，请问这也可以吗。
谢谢大神！

Hello,
I was wondering if this software works with bwa-mem2.
Thanks in advance!

Hello, Dr. Chen,

Recently, I met a problem that after completing the whole pipeline, sometimes there were still lots of "sort.bam.tmp.0000.bam" files left in the output folder, while sometimes they were removed, but I set the same CPU and Mem parameters for each running. I am not sure whether the output vcf file is complete if the bam files are not removed. What might cause this problem and how could I fix it？

Thank you！

Hi,
I am not sure about what is happening here. I use the program on my data (which are non human) and everything seems fine until this step. Then the vcf output file at the end is empty.

Step 3: Validation...

Converting SAM to BAM file, and then Sort and index the BAM file......

[bam_sort_core] merging from 0 files and 11 in-memory blocks...
[bwa_index] Pack FASTA... 0.00 sec
[bwa_index] Construct BWT for the packed sequence...
[E::bwa_idx_load_from_disk] fail to locate the index files
[E::bwa_idx_load_from_disk] fail to locate the index files

Here is my command line to launch the program

perl /home/ubuntu/ERVcaller-1.4/ERVcaller_v1.4.pl -i Dmel_chr2L_sim_100X_150 -f .fq.gz -H /home/ubuntu/genome_Del_1.fasta -T /home/ubuntu/ref_TE.fasta -t 12 -S 20 -BWA_MEM -I /home/ubuntu/ -O /home/ubuntu/Dmel_100X_sim/ -r 150

Thank you in advance for you help!

Hello, Thank you for sharing the ERVcaller.

I was wondering whether this tool can be used for single cell RNAseq data sets (fastq format).

Any suggestions will be appreciated.

hi,
I have human human samples and 2 computers.
I used a same sample on both computers.
but, results were different!
I used same options, programs(samtools, bwa, ERVcaller) version, reference.
additionally, I got error message when sample size over 61G.

readline() on closed filehandle TAB at /home/super/Program/ERVcaller/ERVcaller_v1.4.pl line 1376.

This is my command.
perl $ERV_path/ERVcaller_v1.4.pl -i $samplename -f .sorted.RGfixed.Rmdup.bam -H $refer -T $ERV_path/Database/Human_TE_library.fa -I $input_path/ -O $output_path/$samplename/ -r 150 -t 12 -S 40 -BWA_MEM -G

• I ran multiple samples at the same time each screens.

Hello Dr.Chen,

1.I noticed that the example output.vcf file contained "GQ:GL:DPN:DPI" values, while my output.vcf for each sample only had a result of "GT". What might cause this problem? Any important step I had ignored?

2.When I checked my merge_output.vcf, I found that this vcf file did not contain all the insertions identified in each sample vcf file. I ran over the script of Combine_VCF_files.pl, it seemed like the close insertions would be combined in one region. However, some separate insertions were not in my merge_output.vcf, (for example: Sample1‘s output.vcf contained an insertion on Chr19, while there were no insertions located in Chr19 in merge_output.vcf) ... Why has it happened?

Sorry to bother, thank you so much!!

when @insert_size =0, the script will report errors.
Illegal division by zero at /vol6/home/quluj/zt/software/ERVcaller_v1.4/ERVcaller_v1.4.pl line 1623

Dear Xun Chen,

Hi. I'm Oh.

I have three questions.

1. In your programs, there is no process using "Hydra". Is it right?

2. Where can I find "TE_consensus.fa" in your user manual?

In my ERVcaller-1.4/Database folder, there are three fasta files (ERV_library.fa, HERVK.fa, Human_TE_library.fa).

I want to use all fasta files in database (ERV_library.fa, HERVK.fa, Human_TE_library.fa).

Can I use a merged, and indexed fasta file using three fasta file?

Like this,

$cat ERV_library.fa HERVK.fa Human_TE_library.fa > All.fa
$bwa index All.fa
$perl ERVcaller_v1.4.pl -i TE_seq -f .bam -H hg38.fa -T All.fa

Thanks.

Hi there,

From the improper reads step, bwa-mem fails to read the index files - I did index both the input bam and reference genome fa files, which is why the previous step 'Chimeric and split reads' worked. Beyond that, bwa-mem does not want to work.

Any help would be much appreciated!

Hello, Dr. Xun Chen,
I have a question about the INFO column. For the "INFOR" field, the description stands for "NAME,START,END,LEN,DIRECTION,STATUS". However, it seems that the "START" is not always 1, here is some examples of my results:

#CHROM POS ID REF ALT QUAL FILTER INFO
chr01 629412 . T <INS_MEI:TE_00011020_LTR#ClassII_DNA_Mutator_nMITE> . PASS CR=114;GR=0,0.656,0.745,0.633,0.659,0.444,0.333;GTF=YES;INFOR=TE_00011020_LTR#DNA/Mutator,571,839,269,+,5;SR=8
chr01 951226 . G <INS_MEI:TE_00005384#ClassII_DNA_hAT_MITE> . PASS CR=23;GR=0,0.581,0.359;GTF=YES;INFOR=TE_00005384#DNA/hAT,3526,6274,2749,+,5;SR=9
chr01 951675 . T <INS_MEI:TE_00003559_INT#ClassI_LTR_Copia> . PASS CR=5;GR=0,0.556,0.467;GTF=YES;INFOR=TE_00003559_INT#LTR/Copia,1836,1908,73,-,5;SR=7
chr01 986317 . A <INS_MEI:TE_00006433_LTR#ClassI_LTR_Gypsy> . PASS CR=9;GR=0,1,0.444,0.5;GTF=YES;INFOR=TE_00006433_LTR#LTR/Gypsy,6,2539,2534,+,5;SR=6
chr01 990068 . A <INS_MEI:TE_00011590_LTR#ClassI_LTR_Gypsy> . PASS CR=8;GR=0,0.611;GTF=YES;INFOR=TE_00011590_LTR#LTR/Gypsy,2024,2437,414,+,5;SR=3

I'm wondering what's the insertion range of a TE insertion. For example, in the first record, the POS is chr01:629412, while the "START" of "INFOR" field is 571, does that mean the actual start position for the insertion is "629412 + 571" and the end position is "629412 + 839" ? Or the start position is 629412? In my current study, I need to obtain the start and end postion of TE insertions. I'm confused and could you do me a favor?
Best wishes,
Rain

Hello,

I runned ERVcaller and I found this problem:

Step 2: Detecting TE insertions...

~~~~~ the input bam file was indexed
sh: extractSoftclipped: command not found

What shoul I do?
Thank you.

After I typed the command $perl ERVcaller_v1.4.pl, I got the help message and examples, but I found the examples is conflicting with User Manual of ERVcaller v1.4
The help message is as follows:
perl ./ERVcaller_v1.4.pl

Examples for detecting ERV and other TE insertions:

Detecting TE insertions with a BAM file as the input

   perl /vol6/home/quluj/zt/software/ERVcaller_v1.4/ERVcaller_v1.4.pl -i TE_seq.fa -f .bam -H hg38.fa -T TE_consensus.fa -I folder_of_input_data -O folder_for_output_files -t 12 -S 20 -BWA_MEM

Detecting TE insertions with paired-end FASTQ file as the input

   perl /vol6/home/quluj/zt/software/ERVcaller_v1.4/ERVcaller_v1.4.pl -i TE_seq.fa -f .fq.gz -H hg38.fa -T TE_consensus.fa -I folder_of_input_data -O folder_for_output_files -t 12 -S 20 -BWA_MEM

Detecting TE insertions with separated BAM file(s) as the input

   perl /vol6/home/quluj/zt/software/ERVcaller_v1.4/ERVcaller_v1.4.pl -i TE_seq.fa -f .list -H hg38.fa -T TE_consensus.fa -I folder_of_input_data -O folder_for_output_files -t 12 -S 20 -BWA_MEM -m

Detecting and genotyping TE insertions with a BAM file as the input

   perl /vol6/home/quluj/zt/software/ERVcaller_v1.4/ERVcaller_v1.4.pl -i TE_seq.fa -f .bam -H hg38.fa -T TE_consensus.fa -I folder_of_input_data -O folder_for_output_files -t 12 -S 20 -BWA_MEM -G