FelixKrueger
commented
6 years ago Hi @gbnci,
I just looked and I have downloaded the Opossum genome in 2015 (build BROAD05) already, and it produced the following 7 files in both the CT and GA genome conversion folders:
-rw-rw-r-- 1 fkrueger bioinf 1172288552 Jun 16 2015 BS_CT.1.bt2
-rw-rw-r-- 1 fkrueger bioinf 875415080 Jun 16 2015 BS_CT.2.bt2
-rw-rw-r-- 1 fkrueger bioinf 655316 Jun 16 2015 BS_CT.3.bt2
-rw-rw-r-- 1 fkrueger bioinf 875415075 Jun 16 2015 BS_CT.4.bt2
-rw-rw-r-- 1 fkrueger bioinf 1172288552 Jun 16 2015 BS_CT.rev.1.bt2
-rw-rw-r-- 1 fkrueger bioinf 875415080 Jun 16 2015 BS_CT.rev.2.bt2
-rw-rw-r-- 1 fkrueger bioinf 3665725775 Jun 16 2015 genome_mfa.CT_conversion.faSo you definitely need 6 files ending in .bt2. If I would have to guess I would suspect that you
a) didn't wait long enough for the indexing to complete (judging by the genome size of 3.6GB I would expect it to take between 2 and 4 hours), or
b) that you didn't give it enough memory to work with? Parallel indexing will probably take at least 9GB or more RAM.
Is either of those possible?
I just downloaded the genome you mentioned (Monodelphis_domestica.monDom5.dna.toplevel.fa) from Ensembl (couldn't find that very file from the Broad website), and am indexing as we speak. I'll update tomorrow if it didn't complete successfully for any reason. So far it seems to have started fine:
Bismark Genome Preparation - Step II: Bisulfite converting reference genome
conversions performed:
chromosome C->T G->A
1 138636207 138674540
2 100127924 100132406
3 95991212 96049612
4 79638925 79580201
5 55208532 55150288
6 54020638 54008284
7 46697639 46721056
8 57496204 57378070
X 14957212 14986487
MT 3776 2201
Un 19434538 19520964
Total number of conversions performed:
C->T: 662212807
G->A: 662204109
gbnci
I have downloaded Possum genome sequence from Broad ("Monodelphis_domestica.monDom5.dna.toplevel.fa") and ran genome preparation command as below: "bismark_genome_preparation --bowtie2 . " with "fa" file in the current directory. From the resulting folder "Bisulfite_Genome", two directories were created ("CT_conversion" and "GA_conversion"), but under each one only five files were generated ("BS_CT.1.bt2 BS_CT.2.bt2 BS_CT.3.bt2 BS_CT.4.bt2 genome_mfa.CT_conversion.fa" for CT_conversion and "BS_GA.1.bt2 BS_GA.2.bt2 BS_GA.3.bt2 BS_GA.4.bt2 genome_mfa.GA_conversion.fa" for GA_conversion. While running bismark alignment, I got the following error: Alignments will be written out in BAM format. Samtools found here: '/usr/local/apps/samtools/1.6/bin/samtools' Reference genome folder provided is /scratch/wangyong/possum/ (absolute path is '/spin1/scratch/wangyong/possum/)' The Bowtie 2 index of the C->T converted genome seems to be faulty or non-existant ('BS_CT.rev.1.bt2'). Please run the bismark_genome_preparat ion before running Bismark The Bowtie 2 index of the C->T converted genome seems to be faulty or non-existant ('BS_CT.rev.2.bt2'). Please run the bismark_genome_preparat ion before running Bismark The Bowtie 2 index of the G->A converted genome seems to be faulty or non-existant ('BS_GA.rev.1.bt2'). Please run bismark_genome_preparation before running Bismark The Bowtie 2 index of the G->A converted genome seems to be faulty or non-existant ('BS_GA.rev.2.bt2'). Please run bismark_genome_preparation before running Bismark
Couldn't find a traditional small Bowtie 2 index for the genome specified (ending in .bt2). Now searching for a large index instead...... Seems to me I failed to create the genome necessary for the alignment, could you please give me any suggestions about this. I am using bismark 0.19.0 and bowtie 2-2.3.4 Thanks