genomeassembly-pacbiohifi

I completed analysis and benchmarking of the pacbiohifi for the genome assembly coming from the PacBioHifi reads for the genotypes and haplotypes and their quality assessment. This is the complete genome assembly standardization coming from the PacBioHifi reads and includes all the scripts and the analysis for the reproducibility and the visualziation and the summary analysis.

Genome assembly packages used for the assembly

• hifiasm
• Meryl
• Merqury
• Verkko

Genome assembly evaluation

• QUAST
• BUSCO
• compleasm

Genome Analysis and Project Summary

• The project is loated at the following links given below:
• Project parameters: Project parameters
• Genome assembly summary: Summary evaluation
• Reference evaluation: Reference evaluation
• Installation and configuration: Installation

Haplotype based assembly

• ERR10930361.fastq - maternal
• ERR10930362.fastq - paternal
• ERR10930363.fastq -child
• The hifiasm generates two files when you do the trio binning based on the maternal and the paternal and the filenames outputted are maternalpaternal.asm.dip.hap1.p_ctg.fa and maternalpaternal.asm.dip.hap2.p_ctg.fa and hence they are long so i put the hap1 for the first one and the hap2 for the second one.
• The hifiasm trio binning results are present at trio binning
• All the configuration, run time, wallclock time, nodes, cpu, memory are listed here along with the runtime for each of the analysis project parameters

haplotypes	contigs	Largest contig	Total length	GC (%)	N50	N75	L50	L75
hap 1	823	37880845	534683615	35.47	15838561	9224513	12	23
hap 2	116	31333120	502852466	35.28	23599708	18852907	10	16

• BUSCO evaluation of both the haplotypes: completeness based on BUSCO for the trio binning method ( Total BUSCO searched 425)

Species	Complete BUSCO	Complete and single-copy BUSCO	Complete and duplicated BUSCOs (D)	Fragmented BUSCOs (F)	Missing BUSCOs (M)	Completion score
hap1	422	415	7	1	2	99.2%
hap2	420	407	13	4	1	98.9%

Haplotype assembly without the trio method:

• The last l3 means that for the same sample, the -l3 extensive purging was involved.

contigs	ERR10930361	ERR10930362	ERR10930363	ERR10930364	ERR10930364_l3
	1248	2593	2611	1640	312

---

• since the name of the files after the ragtag are so long so i have named them like this for the easier read:
  • maternalpaternal.asm.dip.hap2.p_ctg.fa.ragtag.correct.scaffold: ragtag1
  • maternalpaternal.asm.dip.hap1.p_ctg.ragtag.correct.scaffold: ragtag2

Ragtag assembly stats on the tribinning asm methods.

haplotype name	placed_sequences	placed_bp	unplaced_sequences	unplaced_bp	gap_bp	gap_sequences
ragtag1	293	473623733	1619	61059882	27100	271
ragtag2	302	465201572	1054	37650894	28100	28

Quast results after the scaffolding with the ragtag.

• The number of the scaffolds have increased and this might be due to the fact that the genome is polyploid so the scaffold have been placed accordingly. The assembled size is near to the grapevine genome.

haplotypes	contigs	Largest contig	Total length	GC (%)	N50	N75	L50	L75
ragtag1	1626	37075411	534706754	35.47	24081286	20838642	10	16
ragtag2	1050	35080446	502871947	35.28	23258691	21132941	10	15

BUSCO report for the ragtag corrected scaffolds.

Species	Complete BUSCO	Complete and single-copy BUSCO	Complete and duplicated BUSCOs (D)	Fragmented BUSCOs (F)	Missing BUSCOs (M)	Completion score
ragtag1	422	415	7	1	2	99.2%
ragtag2	421	408	13	3	1	99.1%

Verkko assembly has been finished, parameters added to the parameters, including the wallclock time, resources used, time of execution, summary below: kmer used 30

• assembly stats of the verkko assemblies using the trio binning. kmer used 30

haplotypes	contigs	Largest contig	Total length	GC (%)	N50	N75	L50	L75
verkko hap 1	2259	33896910	508997582	35.56	10831024	5617093	16	31
verkko hap 2	995	26795713	477196271	35.13	13168430	7656591	13	23

• assembly stats of the verkko assemblies using the trio binning. kmer used 50

haplotypes	contigs	Largest contig	Total length	GC (%)	N50	N75	L50	L75
verkko hap 1	2511	33896910	524953580	35.53	10586138	5279585	17	34
verkko hap 2	1061	26317445	485115873	35.14	13168430	7577555	13	24

• BUSCO results from the verkko assemblies using the trio binning. kmer used 30

assembly type	complete busco	complete and single copy busco	complete and duplicated busco	fragmented busco	missing busco	completion score
hap1	393	385	8	1	31	92.5%
hap2	394	384	10	3	28	92.8%

• BUSCO results from the verkko assemblies using the trio binning. kmer used 50
• By using the high kmer the BUSCO score improved.

assembly type	complete busco	complete and single copy busco	complete and duplicated busco	fragmented busco	missing busco	completion score
hap1	401	392	9	0	24	94.3%
hap2	400	389	11	3	21	94.1%

• assembly mapping reads using the pacbiohifi mapping and the coverage for the haplotypes.

  -- bamstats coverage for hap1 and hap2

  Haplotype	Total reads	Mapped reads	Forward strand	Reverse strand
  hap1	3866068	3863435 (99.9319%)	1936181 (50.0814%)	1929887 (49.9186%)
  hap2	3656794	3653759 (99.917%)	1831768 (50.0922%)	1825026 (49.9078%)

  -- bamstats for the hap1 and hap2

  haplotype	total	unmapped	mapped	mappings ratio	mappings count
  hap1	2330837	2633	2328204	1.65941	3863435
  hap2	2529319	3035	2526284	1.4463	3653759

Genome assembly stats for the hifiasm after polishing the genome with the ERR10930363.fastq reads

• QUAST reports

haplotypes	contigs	Largest contig	Total length	GC (%)	N50	N75	L50	L75
hap 1	2259	33896900	508823952	35.56	10831023	5617076	16	31
hap 2	995	26781654	477006706	35.13	13168428	7656584	13	23

• BUSCO reports of hifiasm polished genome

assembly type	complete busco	complete and single copy busco	complete and duplicated busco	fragmented busco	missing busco	completion score
hap1	422	415	7	1	2	99.2%
hap2	420	407	13	4	1	98.9%

Genome assembly stats for the verkko after polishing the genome with the ERR10930363.fastq reads

• QUAST reports

haplotypes	contigs	Largest contig	Total length	GC (%)	N50	N75	L50	L75
hap 1	823	37880684	534634347	35.47	15838572	9224508	12	23
hap 2	116	31333114	502764077	35.28	23599476	18852818	10	16

-BUSCO reports

assembly type	complete busco	complete and single copy busco	complete and duplicated busco	fragmented busco	missing busco	completion score
hap1	393	385	8	1	31	92.5%
hap2	394	383	11	3	28	92.7%

• I am extending this with the HiC, Chromosomal Application development, Genome bubble solver and other applications.

Gaurav Sablok University of Potsdam Potsdam,Germany