Question

Question: Fragmented ABySS assembly

1

5.7 years ago by

zgayk • 90

United States

zgayk • 90 wrote:

Hello,

We are trying to assemble the genome of the common loon, and I have used abyss (v. 1.5.2) to produce de novo assemblies with the following output for different values of k:

n	n:500	n:N50	min	N80	N50	N20	E-size	max	sum	name	k37
4689853	152662	53975	500	579	761	1177	935	10767	1.19E+08	test-unitigs.fa
4689829	152665	53982	500	579	761	1177	935	10767	1.19E+08	test-contigs.fa
4689653	152727	53871	500	579	762	1178	935	10767	1.19E+08	test-scaffolds.fa
											k55
2564599	25203	9944	500	542	639	867	795	14461	1.70E+07	test-unitigs.fa
2564423	25179	9885	500	543	641	877	799	14461	1.71E+07	test-contigs.fa
2564028	25142	9769	500	543	645	902	811	14461	1.72E+07	test-scaffolds.fa
											k32
5038033	198105	67641	500	591	802	1287	1005	7812	1.61E+08	test-unitigs.fa
5038000	198106	67653	500	591	802	1287	1005	7812	1.61E+08	test-contigs.fa
5037795	198153	67499	500	591	803	1287	1005	7812	1.62E+08	test-scaffolds.fa
											k48
3736945	62667	24079	500	554	678	955	804	9769	4.42E+07	test-unitigs.fa
3736733	62628	24040	500	554	679	961	806	9769	4.43E+07	test-contigs.fa
3735950	62435	23669	500	555	684	986	817	9769	4.45E+07	test-scaffolds.fa
											k64
1636437	5055	1730	500	542	655	1196	1133	12872	3.72E+06	test-unitigs.fa
1636380	5054	1717	500	542	657	1203	1142	12872	3.83E+06	test-contigs.fa
1636124	5088	1698	500	545	669	1282	1159	12872	3.83E+06	test.scaffolds
											k25
6946557	228359	83689	500	578	747	1096	873	5000	1.74E+08	test-unitigs.fa
6946544	228358	83694	500	578	747	1096	873	5000	1.74E+08	test-contigs.fa
6946414	228386	83762	500	578	747	1096	873	5000	1.74E+08	test.scaffolds
											k31
5114778	207133	70364	500	593	809	1301	1015	7999	1.70E+08	test-unitigs.fa
5114751	207137	70181	500	593	810	1301	1015	7999	1.70E+08	test-contigs.fa
5114566	207200	70239	500	593	810	1302	1015	7999	1.70E+08	test.scaffolds
											k30
5192389	216073	73119	500	595	814	1312	1022	7998	1.78E+08	test-unitigs.fa
5192361	216073	73130	500	595	814	1312	1022	7998	1.78E+08	test-contigs.fa
5192194	216128	72984	500	595	814	1313	1022	7998	1.78E+08	test.scaffolds

For the assembly with the highest N50 (814 bp), the contigs are small and highly fragmented (and essentially no scaffolds are produced) even after mapping these contigs to the available red-throated loon genome:

Minimum    Number     Number     Total    Total    Scaffold
Scaffold   of     of     Scaffold     Contig     Contig
Length     Scaffolds    Contigs    Length     Length     Coverage
--------   --------------   --------------   --------------   --------------   --------
All    5,237,924   5,238,436   767,438,425   767,326,331   99.99%
50    3,616,441   3,616,953   710,236,525   710,124,431   99.98%
100    2,146,720   2,147,232   604,271,394   604,159,300   99.98%
250    743,885   744,397   394,016,485   393,904,391   99.97%
500    247,247   247,755   223,350,732   223,238,838   99.95%
1 KB    62,044   62,409   98,533,822   98,431,583   99.90%
2.5 KB    5,725   5,731   18,713,830   18,710,728   99.98%
5 KB    231   231   1,310,589   1,310,589   100.00%

What I am wondering is whether anyone has any ideas why our assembly is so fragmented and if there are any techniques we can use to improve contig length. Sequence data are in the form of pe reads (291,098,878 after filtering) drawn from one insert library size (8kb)? Could the fact that we do not have multiple library sizes be to blame for the small contigs? I do not have an estimate of genome size, but it should be in the range of 1 Gb, and the species is diploid.

Here is the comand I used to run abyss for different k-mer sizes: nohup abyss-pe k=29 name=test29 np=48 in='/share/apps/Data/Loon/COLO1527-8kb_1.filtered.fastq.gz /share/apps/Data/Loon/COLO1527-8kb_2.fastq.gz' &

I am really hoping to find a way to improve contig length, but so far I have not found a way to do this or produce viable scaffolds. Thanks very much for any suggestions.

Zach

assembly • 2.2k views

ADD COMMENT • link •

written 5.7 years ago by zgayk • 90

Indeed, if contigs are short, it will be challenging to scaffold using a single large insert library.
In the first table, what were the kmer sizes? Also, is the "sum" column the size of the assembly? Seems low.
Is your sequencing coverage well above 30x?
Perhaps the heterozygosity is high? There are specific assemblers for that, e.g. Platanus. Anyhow, it's always a good idea to try another assembler.

ADD REPLY • link written 5.7 years ago by Rayan Chikhi • 1.5k

Thank you. The k-mer sizes are on the very right hand side of the first table (they range from 25 to 64, but I did not do every in between). In the sum column these are apparently the sum of the lengths of the contigs at least 100 bp in length).

I am not sure about the heterozygosity, but I have not heard of Platanus. Thanks for that although, I can't imagine why the heterozygosity is higher than other bird genomes assembled using abyss or SOAPdenovo.

The company that did the sequencing for us did an initial SOAPdenovo assembly and we moved on to abyss since their assembly had a contig N50 of approximately 200. So at 814 we have improved with abyss slighly, but not as much as could be hoped.

I believe the read depth was about 33X.

ADD REPLY • link written 5.7 years ago by zgayk • 90

Ah, didn't see the horizontal scrolling bar.. alright.

33x is rather low coverage for de novo assembly. You'd probably get higher contiguity if you sequenced an extra paired-end library.

Follow-up questions:

Did you check for adapters in reads?
How about error correction? See a recent paper (which also reviews other correction tools).

ADD REPLY • link written 5.7 years ago by Rayan Chikhi • 1.5k

I concur; 33x is way too low for 100bp reads on a large diploid genome. With even K=55 you already have only 46 kmers per read, or 33*(46/100)/2 = 7.59x kmer coverage per ploidy, and for a good assembly, you would want an even higher K. You need a lot more coverage for a good assembly, preferably from longer reads (150bp at least; 250bp would be better).

Try to aim for at least ~~15x~~ 20x kmer coverage per ploidy minimum. 30x is better.

Sometimes you can improve things by merging your paired reads, if they are overlapping. That can increase the number of long kmers yielded per pair.

ADD REPLY • link modified 5.7 years ago • written 5.7 years ago by Brian Bushnell ♦ 17k

I think it is great that you've tried many kmer values, but you should also try other assemblers too. I have had great luck with MaSuRCA as compared all other assemblers that I've tested (eg., ALLPATHS N50 was 27kb, SOAP-denovo N50 was 16KB, RAY, I don't want to mention, and MaSuRCA was 2.4Mb!!).

Also, repeat content. What is your genomes estimated repeat content? I know animals have relatively low repeat content, but it will definitely matter!

ADD REPLY • link written 5.7 years ago by arnstrm • 1.8k

Thanks very much to all of you for your help. I really appreciate it. I suspected that our sequencing coverage was too low for a while as many of you have said, but I have just been trying to make the best of the existing data. But your comments have helped me decide that we probably can't improve contig length too much without additional sequencing with another insert library size and higher coverage, so that is what we are going to look into next.

To answer, I do believe that adapters were removed from reads, and error correction has been done, although I was not familiar with BFC. That might be something to use if we get another paired-end library. I am pretty new to this type of work so I haven't estimated repeat content, but most bird genomes have low repeat content for amniotes.

Abyss has worked well for us compared to SOAPdenovo, but these are the only assemblers we have tried. I'll look into MaSuRCA. Any suggestions on what an appropriate insert size for another library would be?

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