An assembly of reads, contigs and scaffolds

A blog on all things newbler and beyond

Posts Tagged ‘scaffold’

Newbler output V: the 454ContigScaffolds.txt and 454ScaffoldContigs.fna

Posted by lexnederbragt on July 12, 2011

Filling the gaps (picture from http://www.ifaonline.co.uk)

In the post on what is new in newbler version 2.6, I introduced the -scaffold option. Briefly, with this option instances (i.e. the consensus sequence) of repeats are placed in gaps. As I mentioned, setting -scaffold results in two extra files. With this post, I will explain these in detail.

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Posted in How it works, Newbler output | Tagged: , , , , , , | 1 Comment »

What is new in newbler 2.6

Posted by lexnederbragt on July 12, 2011

The latest version of newbler, version 2.6, has some welcome additions for input and output. As I have so far only treated de novo assembly, I will skip the updates on the gsMapper (except for mentioning that it is now able to provide a bam file using the -bam option).

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Newbler output III: the 454ContigGraph.txt file

Posted by lexnederbragt on April 13, 2010

The single file I’ll discuss today has in fact almost the entire assembly in it, besides the actual sequences (although even some of these are also included, see below). As explained in my first post, newbler (as many other assembly programs) builds a contig graph. Contigs are the nodes, and reads spanning between them (starting in one contig and continuing or ending in another) indicate the edges. All the information on this graph, except the actual read alignments and consensus contigs, is in the 454ContigGraph.txt file.

The file is divided into several sections, for each one the lines start with a capital letter, except for the first section.

Putting together an assembly...

Section 1) Contig statistics

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Newbler output II: contigs and scaffolds sequence files, and the 454Scaffolds.txt file

Posted by lexnederbragt on March 22, 2010

The files most people are after when they do an assembly must be these: the actual contig and scaffold sequences. The contigs are in the files 454AllContigs and 454LargeContigs. ‘All’ indicates by default contigs of at least 100 bp, while ‘Large’ contigs are at least 500 bp. These lower limits can be set during assembly.
The ‘fna’ files contain the sequences (bases) in fasta format (I actually do not why this extension was chosen over ‘fasta’ or ‘fa’ which are most often used). The ‘qual’ files contain phred-like quality scores (see previous post). The contigs are in the same order between fna and qual files, and the quality scores are in the same order as the bases:

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Newbler output I: the 454NewblerMetrics.txt file

Posted by lexnederbragt on March 11, 2010

With this post, I’ll start going through the output files newbler generates. Some of these will be described in detail as they contain a lot of important information.

For today’s post, we’ll start with the 454NewblerMetrics.txt file. This file contains a lot of details on the reads used during the assembly, as well as the resulting contigs and, in the case of paired end reads, scaffolds.

The file starts with some metadata, such as the date of the assembly, where is is located, and what version of newbler was used. For this post, I used a file of as assembly generated with version 2.3 and both shotgun and paired end read files. Note that the output will be slightly different for a mapping project (to be described in a later post) than for an assembly project.

Section 1: runData

file
{
path = "/your/path/yourfile1.sff";

numberOfReads = ######, ######;
numberOfBases = #########, #########;
}

For each input file, the numbers mentioned are reads and bases in the file, reads and bases after trimming.

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How newbler works

Posted by lexnederbragt on February 9, 2010

I thought to start by explaining briefly how newbler works. I’ll do this by following the output newbler generates during the assembly process. This information is displayed during assembly, and can also be found in the 454NewblerProgress.txt file. It is a good thing anyways to have a look at this file, as it sometimes displays certain warnings (see below).

This example assembly is based on a read dataset consisting of both shotgun reads, and paired end reads (for more on 454 paired end reads, have a look here).

The first thing you’ll see is a message stating that the assembly computation started, and which version of newbler you used.

Then, you’ll see messages for each input file saying Indexing XXXXXXX.sff…, and a counter. During indexing, newbler scans the input file, performs some checks and trims the reads (sometimes more than the base-calling software already did). One of the checks is for possible 3′ and 5′ primers: if a certain percentage of reads contains the same sequence on either the 3′ or 5′ end, this is mentioned. I’ve had some surprises here, such as finding out that reads I got from another group contained an adaptor sequence, which caused problems during the assembly. More on primer removal later…

If an input sff file contains paired end reads, this will be mentioned, as well as the number of reads that contained the paired end linker sequence, for example:

224024 reads, 58599257 bases, 112080 paired reads.

Next:

Setting up long overlap detection…
XXXXX reads to align
Building a tree for YYYYYY seeds…
Computing long overlap alignments…

The first phase of assembly is finding overlap between reads. Newbler splits this phase into one for long reads (this goes very fast) and shorter reads (can take quite some time). As aligning all reads against each other would take too long time, newbler (and many other programs) actually make seeds, 16-mers of each read, where each seed starts 12 bases upstream of the previous one. These seed length and step sizes can be changed if you want (I’ve never tried this, though). When two different reads have identical seeds the program tries to extend the overlap between the reads until the minimum overlap (default 40 bp) with the minimum alignment percentage default 90%) has been reached. These settings can also be changed and influence the alignment stringency, this I will come back to in a later post. Read the rest of this entry »

Posted in How it works | Tagged: , , , , , , | 57 Comments »