Genome Assemblies

Echinoderm Genome Assemblies by Species

Strongylocentrotus purpuratus

Assembly_3.1 (Spur_3.1)

README for Genome Sequence of Strongylocentrotus purpuratus, Spur_v3.1 (June 15th, 2011)

Conditions for Use

The data may be freely downloaded, used in analyses, and repackaged in databases. Some of the data presented here represents work inprogress. It is being released by the Baylor College of Medicine Human Genome Sequencing Center (BCM-HGSC) prior to project completion as a public service to allow our colleagues to search for genes or functions and speed their research. These data have not been edited and are presented "as is." You should regard the data as preliminary if it is unpublished. The data providers and associated funding agencies bear no responsibility for the user's reliance upon or interpretation of these data. The accuracy or reliability of the data is not guaranteed or warranted in any way and the providers disclaim liability of any kind. If you use this preliminary information we request that you honor the following conditions: Please communicate your results to us so that we can incorporate them into the annotation of the final sequence. Contact us at hgsc-help@hgsc.bcm.tmc.edu. Acknowledge the information obtained from BCM-HGSC in publications by stating in Materials and Methods and Acknowledgements: "Preliminary sequence data was obtained from Baylor College of Medicine Human Genome Sequencing Center website." Also acknowledge our funding source, which is listed in each project, with a statement such as "The DNA sequence of [organism] was supported by [grant number from funding agency to PI] at the BCM-HGSC." We also request that you notify us when your manuscript is accepted and send us a pre-print of the article. Use of this data or information derived from it on a web page is permitted, providing the web page contains the statement that "Preliminary sequence data was obtained from the Baylor College of Medicine Human Genome Sequencing Center website." Please inform us of your web page by sending email to hgsc-help@hgsc.bcm.tmc.edu. All other written or oral public disclosures of research using data from the BCM-HGSC should follow the acknowledgment guidelines outlined above. However, although we encourage use of this preliminary information for limited studies, we request that you do not publish whole genome or chromosome scale analyses of genes or genomic data prior to the publication of the BCM-HGSC report on the final genome sequence and analysis. Contact the BCM-HGSC at hgsc-help@hgsc.bcm.tmc.edu to discuss a waiver of this request, which could involve simple acknowledgment, co-authorship, or other methods. Any redistribution of the data should carry this notice.

What's New

This is the eighth release (Spur_3.1) of the genome assembly of sea urchin, Strongylocentrotus purpuratus. This assembly used additional Illumina reads with different end sequence spacing, known as a rainbow library series. The rainbow libraries consist of 4 libraries, a fragment paired end library with ~300bp insert size, and three mate-pair libraries with 1kb, 3kb and 5-6kb inserts. Each library has approximately 10x sequence coverage of genome (see Read statistics below for details). The reads were mapped to the Spur_v2.6 genome assembly and then were used for superscaffolding using the Atlas-Link software and local assembly and gap filling using Atlas-GapFill software. The scaffold N50 increased from 168kb (Spur_v2.6) to 404kb and the contig N50 increased by ~2kb.

Introduction

This is a draft assembly which may contain errors so users should exercise caution. Typical errors in draft genome sequences include misassemblies of repeat sequences, collapses of repeat regions, and artificial duplications in highly polymorphic regions. However base accuracy in contigs is usually very high with most errors near the ends of contigs. A rainbow library sequencing strategy was used to increase the contiguity of the S. purpuratus genome assembly, increasing the average scaffold length and closing assembly gaps. Four different shotgun libraries with nominal insert sizes of ~300bp, 1k, 3k, 5-6kb were constructed for Illumina sequencing. Reads from the recircularized 1k, 3k, 5-6kb libraries were trimmed from the 3' end to different lengths (50bp, 80bp, 120bp) and mapped. Reads that could be mapped were retained at the longest length that mapped to avoid the mapping issues created when the junction fragment is included in the mapped sequence.The reads from the shorter insert paired end library were mapped using the entire read length. After mapping, all reads were combined and used for super-scaffolding and intra-scaffold gap filling with the Atlas-link software. Then a local assembly of reads around each assembly gap was carried out to further fill the gaps using the Atlas-GapFill software. As a result, the scaffold N50increased to 404 Kb and the contig N50 increased by around 2kb. Comparison to the 17,461 available S. purpuratus Unigene sequences from NCBI showed the genome assembly is nearly complete (see the Sequence and Scaffold statistics section below). The number of Unigene contigs aligning 100% of their length increased by 0.1% .

Description of files

The files can be found on the HGSC ftp site ftp://ftp.hgsc.bcm.edu/Spurpuratus/fasta/Spur_v3.1/

• Contigs directory
  • This directory has 3 files for assembled contigs in the genome, there are no chromosome assignments for the contigs in Spur_3.1. The .gz files are compressed with gzip.*Spur_3.1.AGP (AGP file)*Spur_3.1.contig.fa (contig fa)*Spur_3.1.contig.qual (contig quality)*acc_ctg_num.tbl (table listing GenBank accession number for each contig)The AGP file describes how to combine the individual contigs to create the linearized genome sequences in the LinearScaffolds directory.

• Linear Scaffolds directory
  • This directory has 1 fasta file and 1 quality file compressed with gzip.
  • Spur_3.1.linearScaffold.fa (scaffold linear scaffold sequence)
  • Spur_3.1.linearScaffold.qual (scaffold linear scaffold sequence quality)The fasta formatted sequence files are for linearized scaffolds where the gaps between adjacent contigs within a scaffold are filled with 'N's and the captured gap size is estimated from the clone insert size. Each scaffold is a separate sequence within the files.

Sequence and Scaffold Statistics

Assembly	Type	Number	N50 (kb)	Bases+Gaps (Mb)	Bases (Mb)
Spur_v3.0	Scaffolds	31,238	404,330	936,069,451	816,170,552
Spur_v3.0	Contigs	174,512	13,472	816,170,552	816,170,552

Alignment of scaffolds to 17,461 Unigene contigs before and after upgrade

Percent of Scaffolds Aligning to Genome over Alignment Length

Alignment length	100%	95%	80%	50%
Spur_v2.6	89.30%	99.10%	99.80%	99.90%
Spur_v3.0	89.40%	99.20%	99.80%	99.90%

Read Statistics

	PE (300 bp)	1 kb	3 kb	5-6 kb
Total reads	69,680,000	73,200,000	84,453,334	85,480,000
Read length	125bp	150bp	150bp	150bp
Mapped	67.0%	67.2%	68.2%	64.4%
Bridge contigs	3,840,891	6,600,203	9,684,914	10,558,589
Within contigs	32,467,846	21,503,082	20,154,150	14,136,572
Mis-oriented [1]	32,996	41,662	74,554	67,288
Over distance [2]	763,098	430,730	435,184	678,546
Good pairs [3]	31,671,752	21,030,690	19,994,412	13,390,738

[1] Mis-oriented reads map with an orientation of the two ends of the pair that is not expected. For PE, reads are expected to be oriented as -> . [2] Over distance indicates the count of mates with excessive distance between mates, the following insert size cutoff were used:PE: >800bp1k: > 2000bp3k: > 4000bp5-6k: > 8000bp [3] Good pairs refers to pairs in expected orientation and insert size.

Spur_3.1 (June, 2011)Contamination removed version of Spur_3.0.Spur_v3.0 (March, 2011)This release is an improved assembly using a variety of Illumina libraries with different mate-pair distances for scaffolding and gap filling. Spur_v2.6 (April, 2010)Contamination removed version of Spur_v2.5 Spur_v2.5 (February, 2010)Improved assembly of Spur_v2.1 using SOLiD mate pairs.Spur_v2.1 (September, 2006)This release is based on Spur_v2.0, with contaminations removed. Spur_v2.0 (June, 2006) This release is an independent assembly that combines BAC skim readsand WGS reads.Spur_v0.5 (April, 2005) This release update removed 716 contigs of contaminating(non-S. purpuratus) sequence and overlapping (second haplotype contigs).Otherwise the assembly statistics remain unchanged.Spur_v0.4 (March, 2005) ftp://ftp.hgsc.bcm.tmc.edu/pub/data/This release updated the agp file to omit scaffolds of contaminating(non-S. purpuratus) sequence and update coordinates for 65 pairs ofoverlapping contigs. Otherwise the assembly statistics remain unchanged.Spur_v0.3 (November, 2004) This release is the first, preliminary assembly of the California purple sea urchin, Strongylocentrotus purpuratus, genome.

Assembly 2.6 (Spur 2.6)

This version of the updated Strongylocentrotus purpuratus genome sequence was derived from the Spur2.5 version through the removal of contaminating E. coli sequences. The gene sequences have changed very little.

Assembly_2.1 (Spur_2.1)

Spur_2.1 combines BAC reads and WGS reads and utilizes BAC tiling path information. Contaminations identified in Spur_2.0 were removed. Compared to previous assembly releases, Spur_2.1 is more continuous and has fewer false duplications. The Spur_2.1 release was assembled from 2-fold average coverage in sequence reads from Bacterial Artificial Chromosomes (BAC) and 6-fold coverage in Whole Genome Shotgun (WGS) with the HGSC Atlas-2.0 genome assembly system at Baylor College of Medicine. The BAC reads were produced by the Clone-Array Pooled Shotgun Sequencing method (CAPSS) from BAC clones selected based on a minimal FingerPrinted Contigs (FPC) tiling path.In CAPSS pooled BAC reads are assigned to individual BACs by deconvolution. Each BAC assembly was enriched with WGS reads that overlap with the individual BAC reads. The mixed reads sets were assembled locally with Atlas. Sets of overlapping BAC clones were identified based on shared WGS reads and sequence overlaps. The overlapping enriched BACs were then merged together to form the backbone of genome assembly. The merged BAC assemblies were further scaffolded using information from mate pairs, BAC clone vector locations, and BAC tiling path information. Finally contigs from the WGS assembly Spur_0.5 were used to fill gaps in BAC assembly to produce Spur_2.0 release. Extensive contamination analysis was done on Spur_2.0 release. Spur_2.1 release was produced by removing contaminated sequences from Spur_v2.0 release. The Spur_2.1 release includes a set of contigs (continuous blocks of sequence) and scaffolds. Scaffolds include sequence contigs that can be ordered and oriented with respect to each other (multi-contig scaffolds) as well as contigs that could not be linked (single-contig scaffolds or singletons). The N50 of the scaffolds associated with BACs is 216 Kb.The N50 of all scaffolds is 142 Kb. The total length of all contigs greater than 1kb is 804 Mbps. When the gaps between contigs in scaffolds are included, the total pan of the assembly is 907 Mbps. The estimated size of the genome based on the assembly is 814 Mbps.The Spur_2.1 assembly was compared with other available sea urchin sequence data (ESTs, Unigene clusters) to determine the extent of coverage (completeness). A preliminary examination showed over 90% of the sequences in this data set is represented, indicating that the shotgun libraries used to sequence the genome were comprehensive. Typical errors in draft genome sequences include misassemblies of repeat sequences, collapses of repeat regions, and artificial duplications in polymorphic regions. However base accuracy in contigs is usually very high with most errors near the ends of contigs. These data can be downloaded from

ftp://ftp.hgsc.bcm.edu/Spurpuratus/fasta/Spur_v2.1/

Assembly_0.5 (Spur_0.5)

Spur_0.5 is a preliminary assembly of the California purple sea urchin, S. purpuratus, using whole genome shotgun(WGS) reads with the Atlas genome assembly system at the Baylor college of Medicine Human Genome Sequencing Center.The products of the Atlas assembler are a set of contigs and scaffolds. The total length of all contigs greater than 1kb is 768Mb, the N50 of the contigs larger than 1kb is 10.18 kb and the N50 of the scaffolds is 47.98 kb. The total span of the assembly is 1.13 Gb, which is 240 Mb larger than the estimated genome size. The sequence coverage is 6.2X.A preliminary examination showed that over 90% of the sequences in other available sea urchin sequence data sets (Unigene clusters) is represented in the Spur_0.5 assembly. By comparison to 25 NCBI HTGS_PHASE2 BACs( total 2.9Mb), some types of inconsistency were found: several cases of short non-merging overlaps were observed, most at the tail of scalffolded contigs. this may due to polymorphism such that the merging criteria were not met.several short contigs were found aligning in the middle of long alignment gaps of large scaffolded contigs (7 cases), these large gaps come from scaffolding with only short (2 ~6k) and large (50k, 150k)inserts but no middle sized (10 ~ 15 k) inserts, resulting in unfilled large gaps and artificial expansion of total sequence size in the super contigs. Other minor inconsistencies included three cases of differences between genome contigs and PHASE2 BACS, and two possible misjoins. Checking the three contigs in detail did not identify misassemblies. One possible misjoin is in a repeat region and one is a possible local misordering of a short 2k contig in the middle long scaffolded contig.

Patiria miniata

V2.0 Assembly

We sought to improve the Patiria miniata genome assembly with additional PacBio sequences. We generated a new PacBio read dataset at the Duke University Sequencing Center using our reference individual DNA. The read dataset contains 2 million reads and 15.8 billion bp. The read N50 is 10.4 Kb. We used PBJelly2 to combine the PacBio reads with the previously assembled contigs. The results were an improvement in contig size and number with only a small reduction in the number of scaffolds (Table). The P. miniata Gene v2.0 set was generated using MAKER2 pipeline from v2.0 genome assembly.

	Pm v1.0	Pm v2.0
Scaffold number	60,183	57,698
Scaffold N50	52,6141	76,341
Contig number	179,756	131,779
Contig N50	9,466	18,676

V1.0 Assembly

What's New

Pmin_1.0 is the latest (as of Apr 11, 2012) assembly of the genome of Patiria Miniata. The assembly tools CABOG (Celera Assembler), Newbler, ATLAS-Link, and ATLAS-GapFill were used to assemble a combination of 454 reads (fragment and 2.5kb insert paired ends;~15x coverage) and Illumina reads (300bp insert and 2.5kb insert paired ends;~70x coverage).

Introduction

This information is for the first release (Pmin_1.0) of the draft genome sequence of the Patiria miniata . This is a draft sequence and may contain errors so users should exercise caution.Typical errors in draft genome sequences include misassemblies of repeated sequences, collapses of repeated regions, and unmerged overlaps(e.g. due to polymorphisms) creating artificial duplications.

With a goal of solving the polymorphism issues of the data while maintaining the sequence continuity, The Pmin_1.0 assembly was generated in the following steps:

1. 454 reads were assembled by CABOG using settings less strignent than the default (unitigger=bog utgErrorRate=0.03 ovlErrorRate=0.08 cnsErrorRate=0.08 cgwErrorRate=0.14 doExtendClearRanges=0)
2. Both contig and degenerate sequences from the previous step were chopped into fake reads with ~11x coverage (500bp long; 460bp overlap; 80bp minimal length) for ctgs and 8x coverage(450bp long; 400bp overlap; 80bp minimal length) for degs. The fake reads were then assembled by Newbler with the option of -large.
3. Both 454 and iIlumina pair end reads were mapped to the contigs from the previous step. We used BLAT to map the 454 data and bwa(aln+samse) to map the Illumina data, both with the default options. Based on the mapping locations of the paired ends, contigs were then ordered and oriented into scaffolds using ATLAS-Link.
4. ATLAS-GapFill was then used to assemble the reads locally in an attempt to fill the gaps among the contigs within the scaffolds.This final step produced 770.5Mb sequences with contig N50 size of 9.5kb and scaffold N50 size of 50.3kb.

Conditions for use

These data are made available before scientific publication with the following understanding:

• The data may be freely downloaded, used in analyses, and repackaged in databases.

• Users are free to use the data in scientific papers analyzing particular genes and regions if the providers of this data (Baylor College of Medicine Human Genome Sequencing Center) are properly acknowledged. Please cite the BCM-HGSC web site or publications from BCM-HGSC referring to the genome sequence.

• The BCM-HGSC plans to publish the assembly and genomic annotation of the dataset, including large-scale identification of regions of evolutionary conservation and other features.

• This is in accordance with, and with the understandings in the Fort Lauderdale meeting discussing Community Resource Projects and the resulting NHGRI policy statement (http://www.genome.gov/page.cfm?pageID=10506537).

• Any redistribution of the data should carry this notice.

Description of files

There are 2 directories.

1. Contigs/ directory
This directory has 2 files for assembled contigs in the genome, there is no chromosome assignment for the contigs in Pmin_1.0. Pmin_1.0.20120411.contigs.agp (agp file) Pmin_1.0.20120411.contigs.fa (fasta file) The Pmin_1.0.20120411.contigs.agp file describes the positions and orientations of the contigs in the group. It takes the standard NCBI format.
2. LinearScaffolds/ directory
This directory has 1 file Pmin_1.0.20120411.linear.fa The sequences are linearized scaffolds where the gaps between adjacent contigs within a scaffold are filled with 'N's and the captured gap size is estimated from the clone insert size.

Sequence Statistics

Scaffolds/Contigs	Number	N50 (kb)	Bases (Mb)	Gap (Mb)
All Scaffolds	60,336	50.3	811.6	41.1
All Contigs	181,436	9.5	770.5	N/A

History

Pmin_1.0 (Apr, 2012) This release was the first assembly of the Patiria Miniata genome.