Genome comperison showing unaligned sequence of 70kb in synteny

Question

[Umer]

Hi,

I have performed Genome assembly of a fungal sample from Nanopore reads via FLYE-v2.9.5 polishing via Racon an Pilon.

I did a synteny analysis with a closest reference genome and saw some potential chromosomal structural translocations Fig-1. On left are Reference Chromosomes and on right are assembly contigs. As you can see

• Cnt1 with Chr4 and Chr5
• Cnt2 with Chr1 and Chr11
• Cnt6 with Chr4 and Chr5
• Cnt9 with Chr1 and Chr11

Fig-1

the chromosome change in Contig2 and Contig9, there is no unaligned sequence, FIg-2 shwd specific part of contig-2 but contig9 was similar (No-unaligned sequence Gap)

FIG-2

so i was able to create PCR primers arround this alignment shift point and results were +ve. Which meant that this shift is in rality happening in genome.

In contig1 there is an unaligned sequence of ~70kb and in Contig-6 the gap is ~40kb. Fig-3 shows the specific part of Contig-1

Question:

1. How to deal with this issue in genome assembly ?
2. I have extracted that 70kb sequence. How to find this sequence in the RAW FASTQ file. (want to see if this sequence is really there or something else happened during assembly)
3. How can i verify this chromosomal translocation and also this gap-sequence which is coming up in this synteny analysis ? Tried PCR primers but these regions are highly AT-rich so primers didn't work.

Any help in this regard is appreciated.

[GenoMax]

I have extracted that 70kb sequence. How to find this sequence in the RAW FASTQ file.

You can either align your data against this sequence or use bbduk.sh from BBMap suite in filter mode to filter out reads that match the sequence.

[Umer]

Thank you, I will try that. I thee any oither way to verify these contigs ?