Help me debug step 3 in HaploFill

[gluspaula]

Hi,

I need help with troubleshooting HaploFill step 3.
I edited my original post for simplicity because I think I solved my own problem. Nevertheless, for the repeats.bed file, should i include repetitive regions of all .fasta files; hap1, hap2, and Unplaced?

[noecochetel]

Hi,

Sorry for the late answer. Could you please indicate me the file mentioning this HaploFill step 3?
HaploFill usually takes the repeat annotation from hap1 and hap2 concatenated into a single file.

[gluspaula]

Hi, thank you for your response. I figure that part out, but I have a new part I need help troubleshooting.
It's with coverage for HaploFill. I'm having trouble with the ERROR Some sequences failed the coverage information check. I have tried several of the usage combos. -C -s; -C --b1 --b2; and --C1 --C2. I've been trying to troubleshoot the first sequence(contig) that generates the error and the sequence has coverage information for every single base. When I ran -C -s, i was able to see that for this particular sequence, the .bam files don't get generated. I generated the .bam file for this sequence by itself and I get coverage for all bases, plus the bedtools coverage text file with the information as well. One thing i found out is that there are several soft and hard clips in this sequence, so I don't know if this has a role, and I don't know if that is the same or different for the sequences that worked.
Right now I am regenerating the coverage .bam and .txt files using the same minimap, samtools, and bedtools settings that HaploFill.py uses; although, i think this will still not work because I get the same error when i choose -C -s.
Do you have any comments that may help me troubleshoot this step.

For background, the genome assembly.fasta was generated using Flye --keep-haplotypes --meta with ONT reads of a mean qc 18 and min read length of 10k. Final mean coverage in the flye.log was 46x. I used this fasta file for HaploSplit, then I used HaploBreak to break the Ns. Now, I'm inputting in HaploFill.py the following:
python $HaploFill_path
-1 $HAP1
-2 $HAP2
-U $UNPLACED
-c $CORRESPONDENCE
-r $REPEATS
--C1 $COV_HAP1
--C2 $COV_HAP2
-o $OUTPUT_DIR
-t $TEMP_FILE
--map_threads $THREADS
--sequencing_technology ONT

I appreciate any help you can give :)

[noecochetel]

I usually run HaploFill with the following command:

python HaploFill.py -1 ${hap1}.fasta -2 ${hap2}.fasta -U ${unplaced}.fasta -c correspondance.txt --exclusion exclusion.txt -r ${repeats}.gff3 --repeats_format GFF3 -o ${haplofill_run} -C --b1 illumina.on.${hap1}.sorted.bam --b2 illumina.on.${hap2}.sorted.bam > ${haplofill_run}.log 2> ${haplofill_run}.err

Here is an example on how to get the paired-end illumina alignments illumina.on.${hapx}.sorted.bam:

bwa index ${hapx}.fasta 
bwa mem -t 24 ${hapx}.fasta ${reads_1}.fq.gz ${reads_2}.fq.gz | samtools view -bS -T ${hapx}.fasta - | samtools sort -l 9 -@ 24 -m 1500M -o illumina.on.${hapx}.sorted.bam
samtools index illumina.on.${hapx}.sorted.bam

Hope it helps. Please post the content of the error message if you encounter any issues.