To sort your contigs you can use some tools like Contiguator2. But you need a close related reference genome for this tool align your contigs properly, otherwise it will place your contigs in the wrong position.

An option to perform the gap filling is use tools like FGAP or GFinisher that will search for regions in the contigs ends that overlaps and merges these contigs.

To check if it puts the contigs in the correct order you can align your reads to resulting scaffold and verify if there is some region with no coverage.

If you have some difficulties with these tools contact me and I can help you.

I agree with Oliveira that you need closely related reference to make the alignment. Also, i am agree with you that gap-filler tools are questionable.

The best way is to finish your draft genome with additional primer design-and-sequencing at the edge of the contigs. However, the process is laborious and time-consuming but that lead you to make a new reference for the organism.

What you want to do is probably scaffolding, and this, as the previous colleagues said, its better accomplished with a reference genome (same species would be good, otherwise at least the same genus). Another way would be doing more sequencing, for example, using different technology (oxford nanopore or pacbio) or different library preparation method (different insert size or mate-pair kits), and then, use a hybrid assembly method

Thank you for all the answers!

I tried aligning with a closely related species and had success arranging the contigs at least achieving a contiguous sequence, but wasn't sure if it were the right way to do it (in terms of justifying the method in the thesis), because of all the gaps. I wasn't getting anywhere with gap fillers or similar tools. Re-sequencing was not an option.

I am so new to this work and none of my peers know exactly how to guide me, so I appreciate the support!

You may try to assemble your genome with several different tools. Try to use tools with different assembly strategies like SPAdes and Mira. Sometimes a strategy can assemble parts of your genome that another strategy can't. After assembling with different tools you can align them to check if a contig of one assembly can close a gap of another assembly.

Depending on the GC content and some other features of your bacterial chromosome, you can also rely on GC skew plots to order contigs, as origin is usually the portion of the genome with the lowest cumulative GC skew. Check an example here:

https://images.app.goo.gl/xpNpG2KbF5gjxwE77

Of course this is not universal as massive rearrangements due to insertion sequences may also mess with this kind of plot. But it can give you a hint on the ordering.

I know it's been almost 3 years since you made this question, but I'd like to leave a alternative approach that may suits you, or anyone else:

If you have a trusted genome from a close species, you can reorder your contigs by parsing them. Firstly, do this you can use blastn command-line, by setting your contigs as the query and the genome as the subject (Similarly to BLAST interface), you would also set the '-evalue' setting at 1e-10, to avoid any missalignment, and also '-max_hsps' at 1, to ignore the occurence of any indel events that would 'break' your alignment, all of these by using '-outfmt 6' which is the tabular output . Secondly, you can reorder your contigs by using a pipe comand in '|' and sorting your table based in the coordinates of the subject, 'sort -nk 9', and then you can save this table for further purposes, or you can extract only the contigs ID order, by using another pipe with 'cut -f 1', and then extract in the exact position with seqtk or awk.

"blastn -query {your_contigs.fasta} -subject {reference.fasta} -outfmt '6' -evalue 1e-10 -max_hsps 1 | sort -nk 9 | cut -f 1 > ordered_contig_list.txt"

Its worth to notice that this method may return all the contigs that truly scores in the alignment matrix. This method is not a scaffolding method.

Since your supervisor asked you to create a single contiguous sequence for this project... Once you've got the ordered_contig_list.txt file, you can use seqtk or awk to retrieve the sequences in fasta format

"seqtk subseq {your_contigs.fasta} {ordered_contig_list.txt} > {ordered_contigs.fasta}"

Then you can remove all the lines with the '>' ID, and merging into one single sequence with a unique ID. Note again that all the gaps are not going to be reported. So if you wish to perform a annotation step of your contigs, you could do it in the multiple fasta file, and then using BLASTX to retrieve their location in the contiguous sequence.

Best,