# Presentation: 156788

## 16S

- sequences. Find
- near-neighbors.

**Services**

- Trim-away poor quality
- data from a batch of
- sequences.

- Export records from
- the prokMSA.

- Download database,
- presentations,
- and supplemental data.

- Trim

- Align

- Classify

- Classify a queried sequence within a selected database.

- Distance

- Export

- Download

- Calculate a distance matrix.

**Databases**

- 18S

- ITS

- LSU

## Microbial Databases

| Coverage | Region | Records Total | Records Core | Width |
| --- | --- | --- | --- | --- |
| Archaea, Bacteria | 16S | 188,324 | 4,938 | 7,682 |
| Fungi | 18S | 26,930 | 5,201 | 19,663 |
| Fungi | LSU (25/28S -D1D2) | 15,998 | 558 | 2,019 |
| Fungi | ITS | 212,367 | NA | NA |

- Monthly updates

## Database Example: 16S rRNA gene - Total versus Core Set.

- Total
- From Genbank or special collections (Norm Pace, Coral Reefs)
- Keep only 16S genes >= 1,250 bases post-NAST alignment and vector removal - short reads alter tree topology.
- Core Set
- Start by collecting a large set:
- All matching GOLD (genomesonline.org) organisms
*	**     **AND**	*
- All meeting specific criteria
- ≥ 1300 nt.
- ≤ 2 homopolymers
- ≤ 0.3% ambiguity
- ≤ 30 “small gap intrusions” -  bases not supported by the 2° structure.
- Non-chimeric
- De-replicate at >56% common 7mers (~94% similarity).
- 4,938 genes represents the presently known 16S diversity.

- Download either to screen your contigs for 16S content...

## Curation

- Taxonomy
- by independent organizations (JGI, RDP, NCBI, etc.)
- Fungal nomenclature (CBS, MSA, FESIN)
- Core Sets
- Non-cultured novel diversity is only added after similar sequences are reported by multiple studies.

## Error probability: scan and trim

**>actb24**
**TTTTGGGATTCGCTCCGCCTCGCGGCATCGCAGCCCTTTGTACCGGCCAT  TGTAGCACGTGTGCAGCCCAAGACATAAGGGGCATGATGATTTGACGTCG  TCCCCACCTTCCTCCGAGTTGACCCCGGCAGTCTCCTGTGAGTCCCCGAC  ATTACTCGCTGGCAACACAGAACAAGGGTTGCGCTCGTTGCGGGACTTAA  CCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTACAC  CGACCACAAGGGGGCTGATATCTCTACCAGTTTCCGGTGTATGTCAAGCC  TTGGTAAGGTTCTTCGCGTTGCGTCGAATTAAGCCACATGCTCCGCTGCT  TGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAGCCTTGCGGCCGTACTC  CCCAGGCGGGGAACTTAATGCGTTAGCTGCGGCACCGACGACGTGGAATG  TCGCCAACACCTAGTTCCCAACGTTTACGGCGTGGACTACCAGGGTATCT  AATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTAATGGCCCA  GAGATCCGCCTTCGCCACCGGTGTTCCTCCTGATATCTGCGCATTTC.. **
**>actb24**
**..19 10 11 9 8 8 8 15 9 9 10 9 13 8 10 10 10 16 18 16 16   10 9 11 7 8 8 12 14 25 15 15 ****6 6 6 6**** 12 10 14 22 25 21 21 8 10 9 12 11 9 9 17 20 29 29 22 20 11 11 7 7 9 17 13 20 20 31 30 23 23 11 9 9 9 7 7 13 15 25 25 24 21 17 17 17 21 24 24 29 25 25 29 40 32 31 19 19 10 10 9 20 20 25 18 18 25 25 19 19 21 21 23 28 28 29 29 32 22 22 22 32 27 29 25 27 27 22 25 15 15 18 27 27 33 33 33 40 40 47 47 47 32 32 32 32 29 35 40 40 40 40 40 40 31 31 40 32 29 21 21 25 31 26 29 30 30 33 28 31 31 26 26 25 22 22 29 31 28 26 28 27 29 33 25 25 18 27 30 42 37 42 35 35 35 40 40 40 40 42 42 34 34 42 44 47 47 47 47 42 47 42 42 42 42 42 ..**

| Phred | Error probability |  | conf. |
| --- | --- | --- | --- |
| 20 | 1 in 102.0 | 1/100 | 99% |
| 15 | 1 in 101.5 | 1/32 | 97% |
| 10 | 1 in 101.0 | 1/10 | 90% |
|  5 | 1 in 100.5 |  1/3 | 66% |

- qual files should follow sequences throughout pipeline.

## NAST align against the core set.

- Example of NAST (Nearest Alignment Space Termination) compression of a BLAST pair-wise alignment using a 38 character aligned template.  Template and candidate is extended to 40 characters after BLAST gap insertion (A) and retention of original template spacing (B). Nucleotide insertions in the candidate relative to the template which force additional characters to be added in the template are identified at positions  and  (C).  A bi-directional search for the nearest alignment space (hyphen) relative to the insertion terminates at the positions indicated by the black arrows (D).  The leftward search from the  position was shorter in distance compared to the rightward, thus the space left of  ‘GT’ was removed.  The search from the  position encountered the alignment edge on the right, thus the position to the left of ‘AC’ was removed (E). Lastly, the two template-extending spaces are deleted from the template (F).  Notice that sequence data is not added to nor overwritten in the candidate. The NAST removal of two characters from both sequences allowed local misalignments (underlined) while preserving the 38 character format of the global multiple sequence alignment.

- Template: ATAC-----GTA-AC----GTA---C---G-T-AC-GG
- Candidate:CACGTTAAACGTCGTACCCGG

- Template: ATACGT-A-ACGTACGTAC-GG
- Candidate:C-ACGTTAAACGT-CGTACCGG

**(A) pair-wise alignment ****(BLAST/Infernal)**

- Template: ATAC-----GT-A-AC----GTA---C---G-T-AC--GG
- Candidate:C-AC-----GTTAAAC----GT----C---G-T-ACCCGG

**(B) Re-introduce template spacing**

-                                               
- Template: ATAC-----GT-A-AC----GTA---C---G-T-AC--GG
- Candidate:C-AC-----GTTAAAC----GT----C---G-T-ACCCGG

**(C) Identify template-extending insertions**

**(E) Gap at nearest search terminus in**
**       ****candidate removed **

**(F) Gap in template removed **

**NAST**

- Template: ATAC-----GTA-AC----GTA---C---G-T-AC-GG
- Candidate:C-AC----_GT_TAAAC----GT----C---G-T_AC_CCGG

**(D) Search for nearest alignment spaces **
**       ****(hyphens) in candidate**

- retained sequence

- Template sequence

**Finds and extracts 16S portion from any sequence (****tags, reads, contigs or entire genomes****).**

- trimmed

- trimmed

**Candidate sequence**

## Chimera Detection by Bellerophon

- - Enabled by aligning all data to a standard format.
-  Checks libraries internally and against the Core Set.
-  Simple plain-text table delivered.

## Taxonomy in Flux

- Incongruent taxonomic nomenclature even at phylum level.
- Making multiple taxonomic classifications available through Greengenes will aid in standardizing classification, particularly for environmental lineages.
- Greengenes integrates each, allowing a balanced approach to nomenclature of newly discovered organisms.
- EXAMPLE: **Halanaerobium salsugo**
- Hugenholtz=Bacteria; Haloanaerobiales; Haloanaerobiaceae; Haloanaerobium/ Haloincola
- Ludwig=Bacteria; Haloanaerobiales; Haloanaerobiacae; Haloanaerobium
- Pace=Bacteria; Haloanaerobiales; Haloanaerobiaceae; Haloanaerobium/ Haloincola
- RDP=Bacteria; Firmicutes; Clostridia; Halanaerobiales; Halanaerobiaceae; Halanaerobium
- NCBI=Bacteria; Firmicutes; Clostridia; Halanaerobiales; Halanaerobiaceae; Halanaerobium

- Each yellow dot represents a named phyla

- Only a fraction of the phyla are recognized by all five major curators.

## LBL Proposed Standardized Taxonomy 

| Domain | Bacteria | Bacteria |
| --- | --- | --- |
| Phylum | Spirochaetes | Proteobacteria
 |
| Sub-Phylum | Leptospirales_SP | AlphaBetaGammaproteobacteria |
| Class | Leptospirales_CL
 | Alphaproteobacteria_CL |
| Order | Leptospirales
 | Bradyrhizobiales |
| Family | Leptonemaceae | Xanthobacteraceae
 |
| Sub-Family | sfB
 | sfA |
| OTU | 17158 | 12947 |
| Sequence | Leptonema illini str. 3055 | Xanthobacter flavus str. JW/KR-1 |

- The Sub-Phylum containing Leptospirales

- “Rank” initials as helpers.

- >100K full-length high-quality sequences

## Classification Strategy

- Standard
- Apply lane mask.
- Find F84 distance to near-neighbors in each taxonomic system.
- Short reads
- Alignment is not the best approach for short tags from hyper-variable regions.
- K-mer approach is preferred
- de-replicate
- Make confidence-based assignment based on “Observed Node Diversity” empirically derived from region amplified.

## Scalability

- Simple job received by web server, processed by appropriate module of pipeline software and returned by web server.

## Scalability

- Compute intensive job received and staged by web server.
- Grid resource processes job.

## Scalability

- Configuration 1:
- DACC identifies 16S data. Greengenes processes the data according to consortium standards and returns results.

## Scalability

- Configuration 2:
- Individual sequencing centers adopt pipeline software.
- Centers are free to configure thresholds and modify algorithms to suit their hardware.

## Scalability

- Configuration 2:
- Individual sequencing centers adopt pipeline software.
- Centers are free to configure thresholds and modify algorithms to suit their hardware.
- DACC reconciles discrepancies.

## end

## Automation (API)

- REST interface - web service
- Representational State Transfer
- Uniform interface: all resources are accessed with a generic interface (HTTP) using verbs (GET, POST, PUT, DELETE).
- Returns JSON,XML,YAML...

## Intra- and Inter-sample Diversity

## Metadata